8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 1/12

54 9

0026-895X/92 /030549-12$03 .O0 /0

Copyr igh t a by Th e Am erican Soc iety for P harm aco logy an d E xp erim en ta l T he rap eu tic s

Al l r i gh t s of reproduc tion in any form re se rved .

MOLECUL AR PHARMACOLOGY, 41 :549 -560

D rug E fficacy a t G uan ine N uc leo tid e -B ind ing R egu la to ry P ro te in -

L in ked R ecep to rs : The rm odynam ic In te rp re ta tion o f N ega tive

An tagon ism and o f R ecep to r A c tiv ity in the A bsence o f L igand

TOMMASO COSTA , YOSH IO OG INO , PE TER J . M UNSON , H . ONGUN ONARAN , and DAV ID RODBARD

Labora to ry o f T heore tic a l an d P hys ica l B io lo gy , N !C HD (T .C ., V .0 .), an d D iv is ion o f C om pu te r R e searc h and Te chno lo g y (P .J .M ., H .O .O ., D R

N atio n a lln s titu te s o f H ea lth , B e the sda , M a ry lan d 20892

R ece ived Ju ly 29 , 1991 ; A ccep ted D ecem be r 9 , 1991

SUMMARY

The m u tua l e ffe c ts tha t a ho rm ona l lig and (H ) and a guan ine

nuc leo tide regu la to ry p ro te in (G p ro te in ) exe rt on each o the r

w hen s im u ltan eous ly o ccupy ing d is tin c t s ites o f th e re cep to r

m o lecu le (R ) can be v iew ed as the m o lecu la r m echan ism o f d rug

eff ic a cy . These e ffe c ts a re p red ic tab le on th e bas is o f a m ode l

assum ing tha t the te rna ry com p lex be tween the th ree pa rtne rs

(H RG ) reaches equ ilib rium in the m em brane [J . B io l. C hem .

255 :71 08 -71 1 7 (1 980 )]. L igands can be c la ss ified as agon is ts ,

neu tra l an tagon is ts , o r nega tive an tagon is ts , depend ing on

whe th e r they enhance , leave unchanged , o r reduce , re spec tiv e ly ,

the spon taneous tendency o f A to in te rac t w ith G . U s ing th is

m ode l and the assum p tion tha t the G p ro te in response obse rved

in m em b ranes re flec ts th e sum o fig and - independen t (A G ) and

ligand -dependen t (H AG ) recep to r-G p ro te in com p lexes , w e can

exp la in v irtu a lly a ll th e phenom eno logy repo rted ea rlie r fo r op io id

re cep to r-m ed ia ted s tim u la tion o f G TPase , i.e ., 1 ) ex is tence o f

ligands w ith bo th “pos itive ” and “nega tive ” in tr in s ic ac tiv ity (the

la tte r te rm ed n e ga tiv e a n ta g on is ts ), 2 ) equ ipo tency o f neu tra l

an tagon is ts fo r the com pe titive b lo ckade o f the responses e l

ited bo th by agon is ts and by nega tiv e an tagon is ts , and 3 )

pa ren t he te rogene ity o f b ind ing s ite s fo r the b ind ing iso the rm s

o f n eg ativ e an tagon is ts . The te rna ry com p lex m ode l can a lso

exp la in the d iffe ren tia l e ffe c ts o f sod ium on ligand b ind ing a

ligand -dependen t G TP ase ac tiv ity , if w e assum e tha t th is

re d uce s the s ta b ility con s ta n t be tween re cep to r and G p ro te in in

memb rane s . C om pute r s im u la tions p red ic t tha t a nega tive an

tagon is t e xh ib its a d is c repancy be tween “b io log ica l” K (ob ta ined

by S ch ild p lo ts ) and true d issoc ia tion cons tan t fo r the recep to r,

w h ich in c reases as the fra c tion o f “p recoup led ” re cep to rs in t

m em b rane inc reases . T he dem onstra tion o f nega tive an tagon ism

is de fin itive ev idence fo r the ex is tence o f re cep to r coup ling

(hence ac tiv ity ) in the absence o f ligand . U s ing th is expe rim en ta l

pa rad igm , w e show he re tha t spon taneous recep to r ac tiv it

o ccu rs in iso la te d m em b rane s bu t n o t in in tac t N G 18- 1 5 ce lls .

A ffin ity and efficacy are the tw o k ey fac to rs th at de term in e

the in itia l in te rac tion be tw een a recep to r p ro te in and its ligand .

F rom a m olecu lar po in t o f v iew ,ru g e ff icacy ind ica te s the

streng th o f the m icro scop ic change tha t ligand s im part to th e

recep tor m olecu le an d th a t p rop ag a tes to th e g en era tion o f a

b io log ica l s ig na l. P ha rm aco lo g ists h av e deve loped tech n iqu es

to d efine an d m easu re in trin s ic efficacy as a d im ensio n le ss

pa ram e te r based on th e stim u lu s-re spo nse re la tionsh ip o f the

recep to r , ra the r than on its b io chem ical p rope rtie s . T h is co n-

cep t is u n iversa lly ap p licab le to an y recep to r for w h ich a ce llu larre sp onse can b e m easu red (1 -3 ) . H ow eve r , because ou r ab ility

to m an ip u la te th e m olecu la r struc tu re o f recep to rs has b een

en h an ced b y th e u se o f site -d irec ted m u tagen es is tech n iq u es

P resen t address: L ab ora to rio d i F arm aco log ia , Istitu to Sup er io re d i San it#{233} ,

V iale R egin a E lena 299 , 0 0161 Rom a , It lay .

and th e as ses sm en t o f such m an ip u la tions re lie s o n the

chem ica l respon se o f recom b inan t recep to rs tran sfec ted

cells , the re is a c lear need fo r u nde rstand in g and qu an tify ing

e ff icacy in m o lecu lar term s.

F o r som e ty pes o f recep to rs , ligand -b ind in g su bun its

tho se invo lved in th e gene ratio n o f the in trace llu la r sign al

a ll a ssem bled in th e sam e m acrom olecu lan dom ain and , the r

fo re , the d istin ctio n of lig an d-indu ced stim u lus from lig an

gene ra ted b io lo g ica l s igna l is n o t ea sy . In con tra st, G p ro tein

linked recep to rs a re sing le p o lyp ep tide s th at lack the ab ility

gene ra te seco nd m essenge rs (4 ) . T he stim u lus gen era ted by

ligand is tran sm itted to transduce r co up lin g pro tein s (G p

te in s) (5 ), w h ich , in tu rn , transla te and d istribu te it to a

of e ffecto r m olecu les endow ed w ith the ab ility to gene rat

in trace llu la r sign als . T hus , fo r the se recep to rs the degree

ac tiv atio n of G pro te ins is a b io ch em ica l m easu re o f b o th t

ABBREV IAT IO N S : G p ro te in s , guan ine nuc leo tide -b ind ing regu la to ry p ro te in s ; G and G 0 and 3-y subun its o f the guan ine nuc leo tide -b ind ing

p ro te in he te ro trim er; E Cse , e ffe c tive concen tra tion induc ing 50% o f response ; DAD LE , [D -A la2 ,D -Leu5 lenkepha lin ; lC Il 74 , [N ,N ’-d ia lly l-Tyr1 ,A ib2 -

Leu -e nkepha lin ; EG TA , e thy le ne g lyco l b is (fl-am inoe th y l e th e r)-N ,N ,N ’,N ‘-te tra a ce tic a c id ; H EP ES , 4 -(2 -h yd rox ye th y l)-1 -p ipe raz in ee tha nesu lfo n ic

ac id .

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 2/12

K [HR ]- [H ][R] (1)

M ERG]- [R ][G ] (2)

[HRG][R]

( = [HR][RG] (3)

and equations for conservation of mass:

R, = ER] + [HR ] + ERG] + [H RG]

G , = [G ] + ERG] + [H RG]

H , = [H ] + [HR] + [H RG]

The “coupling” constant a, which is both G protein- and ligand-related,

indicates at what extent and to which direction the spontaneous

interaction between R and G is perturbed after the introduction

a is related to the free-energy coupling between H and G , i.e.,

difference between the sum of free energy changes associated w ith

bimolecular reactions (H + R HR and G + Rp RG) and the

standard free energy of formation of the biliganded species (G +

-5 HRG and H + RG -+ HRG) (6) . D epending on whether this energy

of interaction is equal to, larger than, or smaller than zero, ther

“neutral ity,” “antagonism,” or “cooperation” between the binding

and G , which means that the binding of H may leave unchanged

decrease, or enhance, respectively, the probability of R to interact w

G (and vice versa). Because signal transduction depends on the int

action of R w ith G, the existence of a positive interaction (free en

o f co upl ing < 0, a > 1) constitutes a molecular stimulus (the ligand

facili tates the binding of the ligand G). I n pharmacological terms, this

indicates that H has agonistic activity, the extent of which corresponds

to the value of free energy coupling between H and G . In contrast,

the presence of either null or negative interactions (free energy couplin

0, a 1 or a < 1), the ligand H does not facilitate or even deters

binding of R to G . Because such ligands can compete with agonists

the binding center of the receptor, they both can act as antagonists,

and distinction can be made between the neutral and the negati

kinds. T hus, drug efficacy is a molecular property of the receptor

both its partners, hormonal ligand and G protein. The process of sig

transduction at the molecular level can be viewed and quantified as

result of energy transduction within this complex of interacting prot

molecules (6).

The ternary complex model is a minimal representation of the actu

interactions taking place in the membrane. The heterotrimeric G

protein itself is a functional dimer, which consists of a and /3-y subunit

establishing multiple equilibria involving receptor and guanine nucl

tides (5) . T hus, a complete thermodynamic description of the process

should depict energetic interactions between two small ligands

mone and guanine nucleotide) via three distinct protein domains

ceptor, G, and G8 and obviously involves a much greater number

parameters and computations. Extensions of the model that explicitl

consider nucleotide binding have been proposed recently, using e

equilibrium conditions (18) or a steady state approach (19). Even

versions, however, do not address the dissociation of fl-y. I t ispossible to uti lize the simpler ternary complex model to demonstrate

the relation between free energy coupling and ligand efficacy an

predict the differences between antagonists, as long as a number

reasonable assumptions on the relation between receptor-G pro

binding and GTPase response are made, as follows.

I n an experimental context, such as that of biochemical studies

isolated membranes, two forms of output responses can be measure

after addition of the ligand. One is ligand binding, i.e., the sum of

receptor species bound to ligand, [H RG] + [H R]. A second is the exte

of G protein activation, which is reflected in an increase of steadytate

GTP hydrolysis. Studies on the intrinsic fluorescence of purif ied

protein subunits (20) or on the GTP hydrolysis of receptor-G pro

5 5 0 C o s t a e t a! .

1 The nomenclature used here is a combination of those used by D eLean et al.

(7 ) and by Ehlert (17).

and intensity of ligand-induced stimuli before their amplifica-

tion and conversion into intracellular signals.

From a thermodynamic standpoint, the interaction between

hormone, receptor, and G protein can be viewed as a case of

simultaneous binding of multiple ligands (hormone and

protein) to distinct sites of a single monomeric protein (the

receptor). T he energetics of ligand binding at multiple sites of

proteins were reviewed by W eber (6). Based on W eber’s for-

malism, D eLean e t a l. (7) proposed a model of receptor actionknown as the ternary complex model, which assumes that the

stability constant that governs the association between receptor

and G protein can be altered in an allosteric fashion by their

respective ligands (i.e., agonist for the receptor and guanine

nucleotide for the G protein).

An implication of the model is that “efficacy” can be defined

in thermodynamic terms as the mutual effects hormone (H )

and G protein (G ) exert on the binding ofeach other to receptor

(R ) (6, 7) . I f there is a strong positive interaction, the ligand H

facilitates binding of G to R. I n classical pharmacological terms,

this ligand is an agonist; the greater its ef ficacy, the greater its

ability to stabilize the ternary complex. I f there is no interac-

tion, the ligand H does not alter the spontaneous tendency of

R to associate to G and, therefore, is an antagonist that bindsto the receptor site but induces no stimulus. However, the

model also predicts an additional type of antagonist, the bind-

ing of which opposes that of the G protein (and vice versa) and,

thus, promotes destabilization of the ternary complex. I ndirect

evidence for the existence of this type of antagonist comes from

the observation that guanine nucleotides can enhance the ap-

parent binding affinity of some antagonists (8-10). M ore direct

experimental evidence for the existence of antagonists that

destabilize receptor-G protein complexes formed in the absence

of ligand has been obtained in both reconstituted systems (11,

12) and native membranes (13-16). I n this study we have

examined in detail the theoretical aspects of negative antago-

nism predicted by the ternary complex model.

Ma te r ia ls a n d M e t h o d s

Th e Mo d e l a n d It s A s s u m p t io n s

The model consists ofthree components, hormone H , which interacts

at a specific recognition site on the receptor R, and transducer protein

G, which interacts w ith R at a site dif ferent from that occupied by H .

I t is assumed that the interaction reaches equilibrium in the membrane,

so that it can be described as multiple simultaneous binding equilibria

between two ligands and a protein (6-8, 17). T he following scheme

applies:

K

H +R+G = tHR+G

M u 1LaM

H +RG =HRGcK

where K and M are the unconditional equilibrium affinity constants

describing the formation ofthe binary species HR and RG , respectively;

cxK and aM are conditional equilibrium affinity constants for the

formation of the ternary species H RG.’ A s pointed out previously6,

7, 17), thermodynamic balance implies that the ratios between each

pair of conditional and unconditional constants are equal. Thus, the

model is suf ficiently described by the follow ing equilibria:

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 3/12

Negative Antagonism 5 5 1

complexes reconstituted into liposomes (rev iew ed in Ref.1 ) are con-

sis tent w ith the idea that the rate o f nucleotide ex chang e, but no t the

intrinsic catalytic rate for GTP hydroly sis, is the lim iting step fo r

steady state GTPase activ ity . Thus , receptors allow G prote insub-

units to reach the ir max imal cataly tic rate by increas ing the05 fo r

G T P and acce lerating the k, for GD P, an event that is mechanistically

coupled to receptor-induced disso ciation o ffl-y subunits (5 , 2 0-2 1). This

results in a net reduction of the equilibrium constant fo r the nuc leo tide

and net increase in apparent turnov er number fo r GTP hydroly sis .

Accordingly , a simple w ay to simulate ho rmone-stimulated steady stateGTPase activ ity in a membrane using the ternary complex mode ls to

evaluate the amount o f G prote ins bound to the re ceptor. D ata are

show n and analyzed as the sum of the concentrations o f RG and HRG

complexes that are formed in response to increases of the total c oncen-

tration of the ligand. W e refer to this quantity as G prote in response .

C o m p ut e r S im u la t io ns

In its m ost g eneral fo rm , the ternary complex mode l can describe

the inte rac tion betw een n ligands, m recepto rs, and I G prote ins . For

the sake o f simplic ity , w e hav e implemented a form o f the model that

examines n lig ands and I G prote ins interacting w ith a sing le type of

receptor. This is the most common situatio n encountered in an exper-

im ental se tting , w here an inv estigator uses cel l m embrane s containing

a homogeneous population of receptors that can po tentially interac t

w ith a number of G prote in subtypes and that is studied us ing a varie ty

o f agonis ts and antagonis ts . Input parameters fo r the simulations are

the to tal concentratio ns o f the reactants , i.e ., receptor (R), G pro te ins

( Gd ) , and lig ands (T1), plus the affinity cons tants (K and M 3) and

allosteric fac tors (cs that gov ern the ir interactio n. U nknow n variables

are the concentrations o f all the free and bound spec ies . W hen multiple

lig ands are pre sent, only one is allow ed to vary . To compute the

concentrations o f the bound and free spec ies, the fo llow ing set o f

equations must be so lv ed:

E

- 1 + SUM (R)

U) - SUM(G)1

F T‘ - 1 + SUM(B )

where E, U , and F indicate concentratio ns o f empty re ceptor, [R ],

uncoupled G pro te ins , [G ] and free ligands, [H ], respective ly ;T is the

to tal concentratio n of ligand i, and the te rm s SUM (R) , SUM (G), and

SUM(F) indicate the sum of the bound species normalized to the ir free

concentratio ns, as fo llow s:

SUM(R ) = K1F , + M U + (aKF 1M Ui - i f - I i i

SUM(G)J = M E + (aK1F jM E)

SUM(F ) = KE + (a1KEM jU j-1

To obtain a simultaneous so lutio n of eqs . 4 -6 , w e used a s implified

numerical procedure . First, the equatio ns w ere so lv ed sequential ly ,

using as values for the concentrations of free spec ies the corresponding

concentrations o f to tal species present. N ex t, the computed new values

for the free spec ies w ere replaced in the equations , and a new set o f

so lutions w as obtained. Iterations w ere repeated until the concentration

ofeach free lig and present reached a stable v alue (i.e ., w hen the re lative

difference I - F /FjOd < iO . In the absence o f any lig and,

checks fo r convergence w ere done w ith respect to the concentrations

of uncoupled G prote ins.

D a t a A n a ly s is a n d N o m e n c la tu r e

The results o f the simulations w ere, w hen nece ssary , further

lyzed as experimental data w ould be. B inding iso therms expressed

fraction o f bound ligand (BIT) v ersus concentratio n of to tal ligand and

ligand-mediated G pro tein response (i .e ., [HRG] +RG] versus the

concentratio n o f to tal ligand) w ere analyzed us ing a four-parameter

log istic equation (2 2) in o rder to compute s lope facto rs, EC # { 1 2 8 } or I

and upper and low er horizontal asymtotes o f the curv es . B indi

iso therms w ere also analy zed w ith a mass-ac tio n law model for multip

receptor sites us ing the program LIGAN D (23), in order to evalua

the re lationship betw een the parameters of the ternary complex mod

and the apparent number of classes o f binding sites and assoc iated

affinity constants that w ould be st de scribe the data when analy z

using the “incorre ct” mode l. In some case s the curve s w ere reanalyz ed

after the inc lusion of “experimental error.” This w as generated using

random erro r simulator and inc luded in the curves assuming a constant

percentag e erro r of the independent variable across the w hole range

variation of the dependent variable. The fo llow ing nomenclature

used. Paramete rs of the ternary complex model w ere, the aff inity

constant of H (eq. 1 ) usually expressed as a dissoc iatio n constant, Kd

= 1/K ; M , the affinity constant of G (eq. 2 ); and, the coupling

cons tant (eq. 3 ), also refe rred to as “mo lecular eff icacy .” Paramete rs

derived from data analys is w ereK a p , the apparent dissoc iation constant

o f the lig and computed from bimo lecular mass-action law analys is

binding curves (23); andK, the apparent dissoc iation constant of theantag onist deriv ed from Schild plo ts, = 1O”2.

E x p e r im en t a l P r o c e d u r e s

M at er i al s. [ -y -32P] G T P (6000 C i /m m ol ) w as p u r ch asedrom D u-

Pont N ew England N uclear (B o ston, MA ). A deno sine 3’,5 ’-cyc lic

phosphoric ac id 2 ’-O-succ iny l-3 - [’251 ]iodotyro sine methy l es ter (2000

Ci/mmol) w as from Hazlton Laboratories (V ienna, V A ). N eu

blastoma N G1O8-15 ce lls w ere cultured and maintained acco rding

prev iously published pro cedures (24 ).

Assa y of G T Pase. M em b r an es from NG1O8-1 5 ce lls w ere prepared

(4) as described previous ly and sto red at -70’ until used. D eterm inatio n

of GTP hydrolysis w as perfo rmed as described in a prev ious rep

us ing a sodium-free buffer system (15). B rief ly , the reactio n w as

(5) duc ted at 37 ’ fo r 1 0 mm using 5-1 0 zg o f membrane pro teins in a to tal

vo lume of 10 0 jd, in the presence o f e ither N aCI or KC1, and

(6) arrested by the additio n of 25 0 o f H3P04 . The measurement oextent o f P, released (using charcoal) and the calculation of high affin

GTPase activ ity w ere performed as described (1 5).

Asse s sment of r ecep t or -m ed iat ed i n h i b i t i on of cA M P accu -

m u l a t i o n . NG1O8-15 ce lls w ere harv ested and resuspended i

HEPES-buffered iso tonic so lution of the fo llow ing composition in

mM ): N a-HEPES , 20 ; N aCl, 1 35 ; KC1, 4 .5 ; CaCl2 , 2; M gSO4 , 1; EGT

1; supplemented w ith 0 .1% (w /v) bov ine serum albumin. C ells (

i0 w ere incubated at 3 7’ in the same buffer supplemented w ith

M Ro 20-1 724 , in the pre sence o f opio id agonists and antagonists ,

using a to tal vo lume of 1 00 l . The incubation las ted 30m and was

term inated by the additio n of 1 m l of ice-co ld 0 .1HC1 . The concen-

tration of cAMP w as dete rm ined by radio immunoassay , as described

prev iously (2 5).

R ad i ol igan d b i n d i n g st u d i es. B i n d i n g of op i oi d l i gan d s w aseas-

ured as competitio n for the binding sites labe led by [3H]diprenorphine

(0.25-0.5 nM). The reac tio n w as conducted in a buffer identical to

used fo r GTPase, in the pre sence of eithe r N aC1 or KCI, at 2 2’

mm and w as term inated by filtration. Experimental details hav e b

desc ribed previous ly (15 , 2 6) .

R e s u l t s

C ou p l i n g con st an t a as an i n d ex of m olecu lar ef f i cacy .

Fig . 1 demonstrates the interactio n betw een H , R , an

acco rding to the ternary complex model. To simulate the

ation of an experimental setting , the concentrations of R

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 4/12

a= 1000

F ul l a go ni st

a= 3

P ar tia l a go nis t

a= 1

N eu tra l a nt ag on is t

a=0 .3

P artial nega tive antag onis t

x10 # {176}

2.0

1 5 ‘ A

10 4 ’HRG

0 .5 A \G

0 .0 /‘R -4 -2 0 2 4

2. Ci i I

B

1. 5

O#{149 } 4AG + AG-2 0 2 4

2. C , , I

C

1. 5

HRG +RG G

1. G

0 .5

0.c -4 -2 0 2 4

2 .0

1 .0 ± -. 5 G ___AG + AG

0 .5 H A

0. 0-4 -2 0 2

“‘1

I I G1. 5

HAG + AG1 .0 AG

0 .5 H AG

-- .

-.4 -2 0 2 4

Log([Ligand]/Kd)

552 Costa et a! .

a= 0 .0O1

N eg ativ e a nta go ni st

F ig. 1 . R ela tion between a and ligand e ffica cy. D a ta we re simula ted

according to the te rna ry complex mode l, using the follow ing pa ramete rs:

R =G ,= 0 .2 nM ,M= 1 x 1010M 1, andKd= lO O nM . E achpane l

corresponds t o a l ig a n d o f d i f f e r e n t a va lue , as indica ted. P lotted a re the

concentra tions (mol/lite r) of the following specie s: G , uncoupled G pro-

tom; RG , ligand-independent complex; H RG , te rna ry complex; H RG

R G, G prote in response ( th ic k line s). A bsc is sae , logarithms of the mola r

concentra tion of the ligand norm alized to thed.

G are fixed w h ile tha t o f the ligand (H e) is the m an ipu la ted

exp erim en ta l va riab le. T he sim ula tion s d isp lay ed in a ll pane ls

o f F ig . 1 w ere ob ta ined us in g iden tica l va lues fo r, M, and R = G (see legend to F ig . 1 ), bu t each p ane l co rrespo nds to a

ligand hav in g a d iffe ren t v alu e o f the co up lin g con stan t a . In

each pan el the co ncen tra tion s o f G p ro tein spec ie s ( i.e ., u ncou-

p led , [G ], b ina ry com plex , [RG ], te rna ry com plex , [H RG ], and

the ir sum , ERG +H R G ] ) are p lo tted as a func tio n of the

log arithm of the to tal concen tratio n of ligand norm a lized to

th e ligand ’s d is soc iatio n co nstan t fo r the recep to rHKd). A ll

ligands in duce a red is tribu tion of G pro te in spec ies, the ex ten t

and the d irec tion of w h ich depend s o n the va lue o f a . T he

ligand w ith th e h ig hest co up lin g co nstan t (a = 1000) (F ig . 1A )

is th e m ost e ffic ien t in genera ting ternary com plex a t the

exp en se o f b o th free G and RG com plex . A sdecreases , th e

am ount o f H RG form ed in excess o f the in itia l leve l o f

d im in ishes an d , accord ing ly , the reduc tion o f free G also

com es less p rom inen t (F ig . 1B ). Th e con cen tra tion of lig a

y ie ld ing ha lf-sa tu ra tion o f the G pro te in response (EC 50 of the

so lid lines in F ig . 1 ) is sm alle r than the ligan dd when a is

la rge (F ig . 1A ) and tends to approach thed va lue as a

appro aches 1 . B ecause th e ex ten t o f recep to r-G pro tein com plex

fo rm ation de te rm ines the size of the stim ulus induced ,ligand w ith the h ighes t v alue of a is a fu ll ago n ist, in com parison

w ith those hav ing low er a va lues (partia l agon is ts). Fo r a1,

the am oun t o f H RG form ed as a func tion of the ligand concen

tra tio n is exac tly iden tica l to tha t o f RG th at is d isru p ted

Thus , the ir sum and the co ncen tration of f ree G are n o t a lte r

by the ligand at any concen tra tio n (F ig . 1C ) . W hen a is

than 1 , an o ppos ite s itua tion occurs , becau se th e am ount

H RG fo rm ed is sm alle r than the red uc tio n of RG and

concen tra tio n of free G increases (F ig . 1 , D and E ). T hus , i

cons ide r as ou tp u t response of th e sys tem the sum of G pro te

spec ies in recep to r-bou nd fo rm , it is c lea r th at ligand s fa ll

th ree c lasses , tho se p roduc ing a pos itiv e respo nse , o r agon is t

(a > 1), those pro duc ing no resp onse, o r neu tra l an tagon is ts

= 1), and those tha t inh ib it basa l ac tiv ity , o r nega tive an tago-

n ists (a < 1). T h is is exp la ined by the fac t tha t, a s free ene

of coup ling ranges from positive to nega tiv e va lues , the syste

reaches a po in t (a= 1, i.e ., free energ y of coup ling = 0) where

it isju st as p robab le to find H and G bound to d is tin c t m olecu les

o f R as it is to observe the ir sim ultaneous b ind ing to th e s

m o lecu le o f R (6). T h e recep to r bou nd to an an tag on ist w ith

= 1 is energe tica lly in d istingu ishab le f rom an “em p ty ” recep to r,

and , the re fo re, such an an tagon is t can be defined as n eu tra

N eithe r ab ility to produ ce th e te rn ary com plex no r m agn itu de

o f te rn ary com plex fo rm ation is a c riterio n of agon ism

an tagon ism , because it is th e rela tionsh ip be tw een the ligan

indu ced fo rm ation o f H RG and d isso lu tio n of RG tha t de

m ines the ou tp u t from the sy stem .

C ompetition between ligands having differ ent a

ues. T o fur ther illustr ate how a can be used to define

pharm aco lo g ical co ncep t o f e fficacy , it is ins truc tive to exam ine

the m ann er in w hich m ix tu res o f ligan ds h av in g d iffe ren t va lu

of a w ill com pete w ith each o ther in induc ing recep to r coup lin

to G . T h is s itua tion s im ulates experim en ts in w h ich the

sponse of the recep to r is inves tiga ted us ing m ix tu res o f ligan

of d iffe ren t e fficac ies. F ig . 2A show s the G pro te in respo ns

( [ HRG] + [RG] ) g enera ted in th e presence o f inc reas in g con

cen tra tio ns of a fu ll ago n ist (a1 1000 ) and ex am in ed e ith er

in the ab sence or in the presence of com petin g ligands hav i

iden tical a ffin itie s fo r the recep to r (Kd = 10 nM , in th is ex am -

p le ) bu t d if fe ren t va lues o f a2 , w ith a ll be in g presen t a t conce

tra tio ns (10 0 nM ) 10-fo ld grea ter th an the ir d issoc ia tion

s tan t. A s ex pected , th e con cen tra tion -respon se curve o f the fu ll

agon is t is m od ified in a fash ion tha t depend s on th e coup lin

cons tan t o f the com peting lig an d (a2). T he neu tra l an tagon is t

p ro duces on ly a n ig h tw ard sh ift o f the curve , w h ereas par

agon is ts (1 < a < 1000 ) and nega tiv e an tagon ists (a < 1 )

sh ift the low er asym pto te o f th e cu rve in th e upw ard or do

w ard d irec tion , re spec tive ly . S im ila r e ffects are seen w hen

sam e lig ands are a llow ed to com pete w ith a n ega tive an tagon is t

(a i 0 .0 01) (F ig . 2B ).

T he sh ift in EC 50 indu ced by each com peting ligand in

concen tra tio n -resp onse curve of the com peted ligand is larger

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 5/12

-2 0 2

Lo g ( [Lig a nd l/Kd )

a = 1 0 0 0

a 1

N eg at iv e A nta go nis m 553

(9ccI

+

(9c c

a1 = 1 0 0 0

4

F ig . 2 . Compe t i t i on b e t w e e n lig an ds o f d iffe re nt a va lu e s , in induc ing a

G pro te in re s po n s e . The va ry ing lig a nd H1 is e ith e r a fu ll a g o n is ta

1 0 00 ), (A) o r a ne ga tive a n ta g o n is t (a 0 .0 0 1 ) (B ); bo th ha ve th e s a m e

re c e p to r a ffin ity (K d 1 0 0 nM). Th e curves in the a bs e n c e o f c om pe tin g

lig a n d a re dra w n a sth ick lin es. All o the r curves w e re o b ta ine d in the

p r e s e n c e o f a fixe d c onc en tra tio n ( 1 0 0 nM ) o f com pe tin g lig and H 2Kd =

1 0 nM ), w h ich has d iffe ren t va lu es o f a s in dic a te d . Ordinates, to ta l G

pro te in re spo n s e (H1 RG + H2RG + AG) ; abscissae, no rm aliz ed c onc en -

tra tio n o f th e va ry ing lig a nd . O the r pa ra m e te rs a re R ,G 1 = 0 .2 nM, a n d

M 1 x 1 lite rs /m o l. EC O va lu e s a re s ho w n a sla ck dots in each

curve. B e c a u s e a ll c o m p e ting lig a nd s h a ve th e s a m eKd a nd a re pre se nt

a t a c o nc e n tra tio n 1 0 tim e s g re a te r tha n th e Kd , pe r f e c t compet i t iv e

b e ha v io r w o u ld pre d ic t tha t e a c h lig a n d w o u ld pro d uc e a n ide n tic a l s h ift

in th e c o n c e n tra tio n -re s p o ns e c u rve o f th e a g o n is t. In s te a d , th e s h ift

ind u c e d is g re a te r the la rg e r a is .

the larger the value of a f or the competi ng l igand. I n f act,

unl i ke classi cal receptor theory (1-3), the ternary complex

m odel predicts that the apparent potency of a com peting ligand

depends on both receptor af f i ni ty (K ) and ef f i cacy (a). T hus,

i f tw o antagonists have identi cal af f i ni ti es but di f f erent a

values, the l i gand w i th a < 1 w i l l be alw ay s less potent than

that w i th a 1. T his means that, other things being equal , a

negati v e antagonist i s a less ef f i cient “ agonist blocker” than a

neutral antagonist. This departure f rom classi cal pure compet-

i ti ve behav ior depends on the value of. A s M decreases, the

discrepancy betw een competing potency and l i gand af f i ni ty i s

progressi vel y el im inated (data not show n). B ecause asdi -

mini shes the concentration of RG complexes al so tends to zero,

i t i s clear that the ternary complex model predicts dev iati ons

f rom pure competi ti ve behav ior in antagonists only when the

af f i ni ty between R and G is suf f i cient f or spontaneous (l i gand-

independent) coupl i ng to occur.

Y et, the potency of a neutral antagonist as determ ined by

standard “ nul l methods” remains, i n most cases, a good ap-

prox imation of the true af f i ni ty of thi s l i gand f or the corre-

sponding binding center on the receptor. W e simulated the

abi l i ty of a neutral antagoni st to antagonize the G protei

response induced by ei ther a ful l agoni st or a negati ve antago-

ni st. I n both cases, the curves w ere shi f ted to the ri ght as

concentrati on of neutral antagonist w as increased, in a manner

that cl osely approx imates pure competi t i ve behav ior. I n b

cases, Schi l d anal ysi s of these curves y iel ded slopes close

and pA 2 values corresponding to thed of the antagoni st f or

the combinati on neutral antagoni st versus negativ e antagonist

and only sl i ghtl y di vergent f rom thed f or the pai r neutral

antagoni st versus ful l agoni st.2 T hese resul ts are i n good agre

ment w i th the experimental data w e reported prev iousl y

N G1O8-15 cel l s f or opioi d antagoni sts (14).

E ffect of a on the binding properties of the ligand

Competi ti on i s of ten studied by experiments in w hich m ix tures

of l igands of di f f erent ef f i caci es (one of w hi ch i s radiol abeled)

are exam ined for thei r abi l i ty to bind to the receptor. T he ef f e

of a under these condi ti ons i s best seen w hen l i gands of id

ti cal receptor af f ini ty but di f ferent a values are exam ined for

thei r abi l i ty to compete for the receptor bi nding si te l abeled

a variety of radiol i gands w i th var ious values of* . F i g. 3

demonstrates how the IC50 of each l i gand depends on both

a value of the l i gand and the a* value of the tracer. A l though

al l competing l i gands used in Fig. 3 share identi cal af f i ni ti es,thei r competi t i on i sotherms exhibi t a broad range of IC50 values

the relati v e rati os of w hich change for the tw o tracers u

(Fi g. 3).

W hen these data are analyzed using conventional models

assum ing the ex i stence of i ndependent bi nding si tes, the curve

I-

Lo g (E Lig an d]/Kd )

F ig . 3 . E ffe ct o f a o n th e c o m pe titio n is oth e rm s o f Ilg a nds . A, , G , , a nda re g ive n in F ig . 1 Binding is o th e rm s w e re g e ne ra te d fo r s ix lig a nd s

h av in g id e ntic a l a ffin itie s (K d = 1 00 nM), c om pe tin g w ith tw oradio l igands

( for bo th , Kd 1 nM) p re s e nt a t tra c er c o nc e ntra tio n (1 pM ). E ach pane l

c o rre s p o n ds to a d iffe re n t v a lue o f* fo r th e ra d io lig a n d , a s ind ic a te d.

Th e a va lu e s o f th e c o m pe ting lig a nd s a refrom left to right) 1 0 0 , 1 0 , 3 ,

1 , 0 .3 , a nd 0 .0 1 . Ordinates, fra c tio n o f b o und tra c e r. Abscissa, normal-

iz e d c o nc e ntra tio n o f th e c o m pe ting lig a nd s .

2 We performed several simulat ions of the typeescribed above, to assess

under w hich condi tions the discrepancy betw een apparent pA 2 (f rom Schi ld plo

and true dissociati on constant of a neutral antagonist w ould become

noticeable. W e f ound signi f i cant dev iat ions only w hen/M R, or the antagonist

w as al low ed to compete w i th an agoni st of much higher af f in i ty (data not show n

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 6/12

(9c cI

+

(9c c

Lo g ([Lig an d]/Kd )

2 .C x1 0 0

1 .8

1 .6

1 . 4

1 .2

554 Cos ta e t a! .

f or l i gands hav ing a 1 w ere consi stent w i th mul ti pl e bi nding

si tes. T he apparent af f in i t i es and the proporti ons of the si tes

depended on the parti cular combinati on of tracer/competi tor

used. One example of th is anal ysi s i s show n in T able 1, f rom

w hi ch i t i s cl ear that the apparent number of independent

classes of si tes f ound by the f i tt i ng routine depends in a complex

manner on the rati o of a v alues for tracer and competi ng l igand

(Table 1). T hus, i n the case of G protein-l i nked receptors,

anal ysi s by models i nvol v i ng i ndependent cl asses of si tes may

gi ve i nappropri ate and m isl eading resul ts7, 8, 18) .

E ffect of the magnitude of on receptor -mediated G

pr otein r esponse and ligand binding. H ow does the m ag-

ni tude of the stabi l i ty constant M af f ect the theoretical concen-

trati on- response curves for vari ous l i gands? Fig. 4 show s that,

as M i s vari ed over 6 orders of magni tude, the concentration-

response curves for agoni st- and antagoni st-mediated G protei n

responses undergo complex changes. I t i s convenient to exam -

me these changes as zones w i thi n w hi ch the di ssoci ati on con-

stant 1/M vari es w i th respect to the concentrati on of receptors,

because the interacti on betw een R and G (governed by)

occurs w i thi n the plane of the membrane. Z one 1 corresponds

to an increase of the di ssoci ati on constant betw een receptorand G protein f rom a smal l value to one close to the concentra-

t i on of receptors (i .e., 1/M U nder these condi tions,

the decrease ofM primari l y changes l igand-i ndependent acti v -

i ty w i thout al teri ng the EC50 and the max imal ef f ect of the

concentrati on-response curves of the agoni st (Fi g. 4A ),

w hereas, for the negati ve antagonist (Fig. 4B ), the IC50 values

decrease and max imal ef fects are augmented asdiminishes.

I n Z one 2, when 1/M Rtota i, the EC50 values of the agoni st

are progressi vel y i ncreased (Fig. 4A ), w hereas those of the

negativ e antagonist no l onger change (Fig. 4B ). I n Z one 3,

f urther i ncreases of1/M (i.e., 1/M >> RO5) produce only

changes in the max imal ef f ects of the agoni st (Fi g. 4B ), because

under these condi ti ons ERG] i s negl i gible and l i ttl e or no

spontaneous acti v i ty occurs. T hus, zone 3 descri bes a si tuati on

i n w hi ch coupl ing betw een R and G is absolutely dependent on

TABLE 1

Ana lys is w ith the p rog ram L IGAND o f b in d in g iso the rm s o f ligands

o f d if fe re nt a c o m p e ting w ith a ra d io tra c e r h a v in ga* I

The analyzed curves c o rre s p o nd to th o s e s h ow n inig . 3 B ( tra c e r a 1 ).

Parameters u s e d fo r the s im u la tio n s a re A, c i, = 0 .2 np , K ,, o f c om pe tin g l igands

- 1 0 0 nii, K 5’ o f tra c e r = 1 n (p re s e n t a t 1 pM) , a n d a ll o the r p a ram e te rs a s in

Fig . 3 . D ata w e re a na ly ze d b oth b efo re a nd a fte r th e a dd itio n o f e xp e rim en ta l n o is e ,

as e x p la ine d In Ma te ria ls a n d Me th o d s . Th e p a ra m e te rs ta b u la te d a re tho s e

c om pu t e d from efTor-free data a nd c o rre s p o n d to the m o s t c om p le x m o d e l tha t

cou ld produce a s ig n ific a nt im pro v e m e n t o f the fit (2 3 ). F ifte d pa ra m e te rs a re

apparent dissociation co n s t a n t s ( Km , ) a nd de ns ity o f b ind in g s ite s (A). The le ve l o f

s i gn i f i cance w as t aken a s p = 0 . 0 5 .

PvMe te rs a 1 0 0 ’ a lO a3 a 1 a 0 .3 a0.01

K (nM) I .5 1 4 4 2 1 0 0 2 2 0 7 4 0

K.1 , , (nM) 1 0 4 3 8 4 1 2 0 3 6 0

K .nM ) 71 93 124

R 1 (pM) 1 5 0 1 3 5 1 3 6 2 0 0 1 0 0 2 0

A2 ( pM) 4 0 4 7 6 4 1 0 0 8 8

R 3(pM ) 13 18 91

S i t e s de te c te d with 3 3 2 1 2 3

no errorb

S ite s de te c te d w ith 3 2 1 1 1 2

3 % e rro raa a va lu e o f the c om pe tin g lig an d .

b This ind ic a te s the num b e r o f a p pa re n t b ind in g s ite s tha t w o u ld h a vee e n

in fe rre d fro m th e a na ly s is o f b ind in g c urv e s .

1 .0

0 .8

0 .6

0.4

0 .2

-3 -2 -1 0 1 2 3 4

Lo g (E Lig an d]/Kd)

F ig . 4 . E ffec t o f re du c tio n s o fM o n the G p ro te in re s po ns e . Th e va ry ing

lig a n d is e ithe r a fu ll a g o n is t (a 1 0 0 0 ) (A) o r a ne g a tive a n ta g o n is t (a

= 0 .0 0 1 ) (B ). B o th lig a n ds h a v e th e s a m e a ffin ityK d = 1 0 0 nM); A, =

= 0 .2 n E a c h curve c o rre s p o n d s to a d iffe re n t v a lue o f th e p a ra m e te r

M , wh ic h d e c re a s e s in lo g a rithm ic s te p s o f 0 .2 5 s ta rtin g from th e f

c u rve , a s ind ic ate d.

the presence of the l i gand, and the distincti on betw een neut

and negati v e antagonists i s no longer possible.

T he corresponding ef f ects of vary ing M on the bindi ng of the

l igand are documented in Fig. 5, w here bindi ng i sotherms

ful l agoni st (Fi g. 5A ) and a negati ve antagoni st (F i g. 5B

competi ti on w i th a tracer concentrati on of neutral antagonist

are displayed for several values of/M . A s observed for recep-

tor-mediated acti vati on of G , the decrease of(i .e., i ncr ease

of 1 /M) af f ects the bindi ng of agoni st and negati ve antagoni st

in opposi te di recti ons, and the ef f ect of change ini s most

prom inent in zone 1 for the negati ve antagoni st and in zone

and 3 for the agoni st (compare Fig. 5, A and B ).

I t i s cl ear f rom inspecti on of Fi g. 5 that, the l arger the v

of M , the more the ratio betw een the EC50 andd of the l i gand

di f f ers f rom uni ty (< 1 for agoni sts and >1 for negati ve an

oni sts) . Such departure of EC5O /K d f rom uni ty i s largermore a di f f ers f rom 1 (data not show n). T hus, the reducti on

aff in ity betw een R and G af f ects the concentration-response

curves of l i gands in a di recti on determ ined by , and to an ex

proportional to, the ef f i cacy of the l igand. T he rel ati onship

betw een a l igand’ s EC50 and1/M i s al so i nf l uenced by the

stoi chi ometry betw een R and G W e found a 10-f ol d ri ghtw ard

shi f t i n the rel ati onship betw een change in EC50 for

agoni st and antagoni st and increase of/M w hen there w as a

10-f ol d molar ex cess of G proteins rel ati ve to the concentration

of receptors (data not show n).

I nter pr etation of “sodium effect” in ter ms of equilib-

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 7/12

a * = 1 , a = 1 0 0 0

I-Lo g ([Lig an dl/Kd )

a* 1 , a - 0 . 0 0 1

C

0

0Co.0

+

(D

+

I

B

.

0 .8 BA SA L

0 . 6 _ _ _ _ _ __ _ _ _ __ _ _ _ _

0 .4 cr -O .O1 , c r=O .OO1

-1 0 -9 .5 -9

Log (1 /M)

-3 -2 -1 0 1 2 3 4

Lo g ([Lig an dl/Kd )

-4 -2 0 2 4

Neg a t i v e A n t a g o n i s m 5 5 5

F ig . 5 . E ffe cts o f re duc tio n o f th e s ta b ility c o n s ta n tM on the b in ding

is o th e rm s . Lig a nd is e ith e r a fu ll a g o n is ta 1 0 0 0 ) (A ) o r a ne g a tive

an tagon i s t (a = 0 .0 0 1 ) (B ). P a ram e te rs w e re a s in F ig . 4 . B o th va ry ing

lig a nd s (A a nd B )(K , , = 1 0 0 n M) c o m p e t e fo r th e b ind ing s ite s la b e le d by

a ne utra l a n ta g on is t ra dio iig and (K d = 1 00 pM, a 1 ) p re se nt a t tra c e r

c onc e ntra tio n (1 pM) .

r ium coupling of R and G , i.e., as a r eduction of. The

effect of M on the curves for receptor-mediated activation of G

protein closely resembles the effect of sodium ions on agonist-

mediated stimulation of GTPase activity. U sing opioid recep-

tors in membranes prepared from NG1O8-15 cells, we have

shown previously that there is an inverse relationship between

intrinsic activity of the ligand and the ability of sodium ions to

reduce GTPase in the presence of that ligand (15). H ere we

show that those findings can be simulated assuming that so-

dium ion results in a discrete reduction of. The reduction of

G protein response in the presence of saturating concentrations

of ligand, as a function of, depends on the a value for the

ligand (Fig. GA ). I fwe examine the range of/M values betw een

0.1 and 1 nM (Fig. 6A ), the decreases of G protein responses

ar e linear w ith log(1/M ) (the depar ture fr om a linear r elation

would not be expected to be detectable in the pr esence of

experimental noise) . T he slopes of such lines are measures of

sodium effect (Fig. GB). The slopes are larger as the value of a

approaches 1 and progressively decrease as a becomes either

very large or very small. Thus, the relation between simulated

sodium effect (slopes of the curves in Fig. 6B) andis bell-

shaped (F ig. 6C ) and closely matches the pr eviously r epor ted

relationship between experimental sodium effect and apparent

intrinsic activity of the ligand (see Fig. 5 in Ref. 15). W e did

not observe a similar pattern of decrease of responsiveness

when other parameters of the model (such as reduction ofd

or a of the ligand) were selectively varied (data not shown).

Log (1 /M) Lo g (a)

F ig . 6 . S im u la tio n o fs od ium e ffe c t o n th e G p ro te in re s p o n s e A, P a ram -

e te rs w e re a s in F ig s . 4 a nd 5 . The G pro te in re s po n s e (HAGAG )

c om pu te d a t m a x im a l c o n c e n tra tio n o f iig a n d (1 0 0 0 x EC is p lo tte d

fo r d iffe re nt va lu e s o fog(1 /M). Ea c h curve c o rre s p o n ds to th e re s p o n s e

g e n e ra te d in the p re s e n c e o f a lig a n d o f d iffe re n t a va lu e , a s ind ic ate d.

Th e th ick so lid line c o rre spo nd s to th e ba s al re spo n s e m e a s ure d in th e

a b s e nc e o f lig a nd (wh ic h is id e n tic a l to tha t in th e pre s e n c e o f a lig a n

with a = 1 ). To s im u la te e xpe rim e n ta l da ta , a n a rb itra ry ba c kg ro und o

1 x 10 -1 0 M ha s be e n a dde d to th e s um o f re c e p to r-bo und G pro te in

s p e c ie s a n d it re p re s e n ts th e G TP a s e hyd ro ly s is du e to fre e G . B , T

re du c tio n o f m a xim a l G pro te in re spo n s es o b s erv e d w ith in th e “w indo w ”

o f 1/ M va lu e s o f 0 .1 -1 n (s e e Ahaded a rea) ha s be e n n o rm a liz e d to

th e ba s a l re s po n s e o b s e rv e d a t /M = 0 .1 n in the presence o f a lig a nd

with a = 1 . C , The s lo pe s c o mpu te d from th e c urve s o f B w e re re plo tte d

a s a func tio n o fthe lo g a rithm o f a.

E ffect of sodium on the binding isother ms of opio

ligands, show ing appar ent m ultiple aff inity states.

further explore the relationship between sodium effect

reduction of M , we used a combination of exper imental data

and simulation according to the ternary complex model. Fig.

illustrates how the replacement of potassium by sodium in

reaction affects the binding isotherms of a full opioid agoni

( D A D LE ) and two antagonists, a putative negative antagonist

(1C1174) and a putative neutral antagonist (M R2266), as deter

mined from their effects on GTPase in NG1O8-15 cell me

branes (14, 15). Sodium induced a rightward shift in the isot

erm of the agonist (Fig. 7A ) and a leftward shift in that of

negative antagonist (Fig. 7B). In contrast, the bindingSO-

therms of M R2266 remained unchanged (Fig. 7C ). A nalysi

according to mass action law of these data suggests comple

and divergent effects of sodium on the curves of the agoni

and antagonist. T he binding isotherms for the agonist

consistent w ith three apparent states of different affinities

the absence of sodium, very highK., high (K ap2), and low

(K ap3), with dissociation constants ± standard errors (thr

experiments) of 0.55 ± 0.02 nM , 33 ± 12 nM , and 7.2 ± 2.5

respectively. T he corresponding proportions of sites existing

the three aff inity states are R1, 85 ± 10% ; R 10 ± 5% ; and

5 ± 2% . W hen sodium was replaced by potassium, only tw

affinity states were detectable (K ap1, 1.5 ± 0.3 nM ; Kap 170 ±

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 8/12

x0

Log (T )

10 0

10 0

Neutral

:98:7r65

50

L o g ([U g a n d llK c j)

F ig . 7 . S im ila rity be tw ee n e ffect of sodium on expe rimenta l binding isothe rms (A, B, and C ) and reduction ofon simula ted binding curves (D , E ,

and F ). Uppe r , ligands a re a full agonist (D ADLE ) (A), a puta tive nega tive antagonist ( IC l 174 ) (B), and a puta tive neutra l antagonist (M R2266 )

compe tition for the sites labe led by the pa rtia l agonist [3H ]diprenorphine (250M) in the pre sence of 1 50 m le ve ls of e ither KC1 or N aC l (0 ).

Binding was measured using 230 ,g/m i ce ll m em branes in T ris/H EPES (5 0 m M), p H 7 .5 , conta ining 10 m M gC i2 , 0 .2 m dithiothre itioi, 0 .2 m

EG TA, 50 ,g/m l bacitracin, and e ithe r KC I or N aC I as indica ted. The incuba tion lasted 60 mm at 20# {176 }nd was te rm ina ted by filtra tion. D a

expressed a re pe rcentage of the binding of trace r obse rved in the absence of compe ting ligand. TheIT of the tra ce r was 0 .1 44 and 0 .10 in thepresence of KC I and NaC I, respective ly. T he i nes represent best fit of the da ta according to a mass action law mode l.owe r , sim ula te d co mp etitio n

isotherm s. The radioligand is a partia l a gonista* 0 .3 , Kd ’ = 0 .31 nM , present a t a fixed concentra tion of 0 .25M ); competitors are a full a gonist

(a 300 , Kd 31 6 nM ) (D ), a negative antagonist (a 0 .0 1 , K d = 25 n M) (E ), a nd a neutra l a ntagonist (a 1 , Kd = 2 n M) (F ). E ach pane l shows the

binding curve obta ined a t high (4 x 110 M1 ) and low (2 x iO M1 ) va lue s of the sta bility consta nt M . O the r parame ters a re A, G , = 0. 2 n Data

ar e plotted as inupper , but the absc i ssae show the normaliz ed concentra tion of tota l ligand.

5 5 6 C o s t a e t a! .

10 0

A g o n

(DAD

A

is t

LE)

0 NaCI

0___ #{ 163}

-10 -9

.

-8 -7

I

-6 - 5

50 nM ; R1, 75 ± 6%; R2, 2 5 ± 5%). The curves for the negative

antagonis t indicated tw o affinity s tates in potass ium (L,, 5 40

± 150 nM ; K 60 ± 12 nM; R1, 7 0 ± 10%; R2, 30 ± 4%).

Replacement w ith sodium increased the of the high affinity

state and reduced the propo rtio n of the low affinity s tateKap,,

800 ± 350 nM; Kap2, 20 ± 2 nM ; R1, 8 ± 2 .5%; R2, 92 ± 8%).

The binding iso therm s of MR2266 are consistent w ith a sing le

affinity fo rm of the receptor in both KC1 and N aClKap, 3 .8 ±

0 .4 and 2 .9 ± 0 .5 nM , respective ly ).

S imulated binding iso therms for a full agonist (a300) , a

negative antag onis t (a = 0.01) , and a neutral antagonist (a =

1) in competitio n for the binding of a partial agonist (a3)

w ere g enerated for tw o values o f (Fig. 7 , D , E , and F). A 20-

fo ld reduction o fM alters the binding iso therms of the agonis t

and negative antagonist in a w ay that c lose ly resembles the

e ffec t o f sodium on experimental binding data. W hen the same

data (to w hich random gaussian error w ith 3% coeff ic ient o f

v ariatio n w as added) are analy zed by the program LIGAND ,

the binding curves for the agonist and negative antag onis t (but

no t those fo r the neutral antagonist) are consistent w ith mul-

tiple binding sites, the proportion and aff initie s o f w hich are

altered by a reduction of in a pattern very s im ilar to that

observed for sodium in experimental data.

N egat i v e an t agon i st s as p r ob es f or t h e ex ist en ce of

l i gan d - in d ep en d en t r ecep t or cou p l i n g. B ecau se t h e com -

peting po tency o f a negativ e antagonist is inversely re lated to

the magnitude o fM , it is to be expected that the apparent pA 2

of such antagonists w ould be increased by a dim inution of

and, therefore, by an increase o f sodium concentratio n. Fig . 8A

show s the theo retical S child plo ts for a negative antag onist (a

= 0 .01 ) in competition w ith a full agonist, o btained from

simulated curves g enerated us ing tw o different values o f.

The concentrations o f negative antagonist that produce equ

alent shifts in the concentration-response curves o f the agonis t

are reduced as M dim inishes (Fig . 8A ). Consequently , the

apparent K (10_PA2) of the negativ e antag onist approaches th

true Kd value w hen M becomes smaller (see legend to Fig . 8A )

In Fig . 8B w e show corresponding experimental data obtaine

us ing N G 108- 15 ce ll membranes . Concentration-response

curves for DA D LE-stimulated GTPase activ ity in the presence

of increasing concentratio ns of the neg ative antagonis t 1C1

w ere obtained in e ither N aC1 or KC1. The apparent< value

for 1C1174 w as decreased in the presence of N aCl, w hich

cons is tent w ith the idea that sodium acts by reducing. In

contrast, S child plo ts for MR 2266 w ere v irtually superimpos-

able in N aC1 and KC1 (data not show n). The theoretical S c

plo ts o f the neg ative antagonis t at high values ofexhibit a

slight but clear dev iation from linearity (Fig . 8A ). How eve

this dev iation is too small to be detec table in the presence

experimental error and should no t be used as a diagnostic

fo r negative antag onism . The sodium-induced shift o f the

S child plo t o f the antagonis t is more readily accessible

experimental detec tion and less prone to be o verlooked,

in the presence o f realis tic experimental error.

L ack of sp on t an eou s r ecep t or act i v i t y i n i n t act c

To investig ate w hether spontaneous receptor activ ity , w

c learly ex ists in iso lated membrane preparations , also oc

in intact ce lls, w e studied the behav ior o f the negativ e anta

nis t 1C1174 as a blocker of agonist-mediated inhibition o f

accumulation of cAMP lev els in intact NG1O8-15 ce lls. Exp

iments designed to assess the effec t o f IC I174 on basal cA

accumulation gave inconsistent results. A lthough in some

periments w e observed an antagonis t-induced enhancement of

basal activ ity (22%), the effec t did not exhibit c lear concentra-

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 9/12

0

a)

w

Co

0.4-s

0

CO

U-

-8 .0 -7 .0 -6 .0

Lo g [N e g . An ta g .], M

i o

Lo g [D AD LE ] M

Lo g [IC I 1741 , M

1 0 _ i

-8 -7 -6

N eg at iv e A n tag on is m 557

c c0

0)

.3

F ig . 8 . S c h ild p lo ts o f a n e g a tive a n ta g o n is t: e ffe c t p re d ic te d fo r a

re d u c tio n o f M (A) o r e xp e rim e n ta lly d e te rm in e d fo r a n in c re a s e o f N a

(B). Ordinates, lo g (D A-1 ), w h e re D R is d o e s ra tio , i.e . , th e ra tio o f E C

va lu e s fo r th e c o n c e n tra tio n -re s p o n s e c u rv e s o f th e a g o n is t in th e

p r e s e n c e a n d a b s e n c e o f a n ta g o n is t. Abscissae, lo g a rith m o f th e c o n -

c e ntra tio n o f a nta go n is t. A, C o n c e n tra tio n -re s p o n s e c u rve s s im ila r to

th o s e in F ig . 4 w e re g e n e ra te d in th e a b s e n c e a n d p re s e n c e o f in c re a s in g

c o n c e n tra tio n s o f a n e u tra l a n ta g o n is t. P a ra m e te rs w e re R ,G , = 0 .2

n M; v ar y in g lig a n d ( a go n is t) , a 3 0 0 , Kd 3 1 6 nM ; a nd c o m pe tin g lig a nd

(n eg a tive a n ta go nis t), a 0 . 0 1 , Kd 2 5 n u . Th e tw o p lo ts w e re

g e n e ra te d u s in g d iffe re n t va lu e s o f ( in lit e r/ m o l) , M = 4 x 10 10 ()

a n d M = 1 x 1 0 (- - - ). Th e c o rre s p o n d in g p a ra m e te r e s tim a te s b y

lin e a r re g re s s io n a re K1 = 2 1 3 .4 n a n d s l o p e = 0 .8 7 fo r th e h ig h v a lu e

o f M a n d K, = 3 5 nM a n d s lo p e = 0 .9 9 fo r th e lo w va lu e o fM . No te th a t

th e lo s s o f lin e a rity a t th e h ig h e s t va lu e o f i s m in im a l, a n d it w o u ld b e

d iffic u lt to d e te ct in th e p re s e nc e o f e xp e rim e n ta l e rro r. B , C on c e n tra tio n -

re s p o n s e c u rve s fo r D AD LE -m e d ia te d s tim u la tio n o f G TP a s e a c tiv ity in

N G 1 0 8 -1 5 m e m b ra n e s in th e p re s e n c e o f in c re a s in g c o n c e n tra tio n s o f

IC I1 7 4 w e re g e ne ra te d u s in g a re ac tio n m ix tu re c o n ta in in g 1 50 m le ve ls

o f e ith e r KC I (0 ) o r N a C l (S ) . A fo u r-p a ra m e te r lo g is tic m o d e l (2 2 ) w a s

u s e d to c o m pu te E C va lu e s a nd th e ir re la tive ra tio s ; d a ta w e re re plo tte d

in S c h ild c o o rd in a te s , a n d lines a re th e c o rre s p o n d in g lin e a r re g re s s io n s .

Th e fo llo w in g va lu e s w e re c o m p u te d : in KC I (0 ), p A26 .7 8 ± 0 .1 5 (K,,

1 67 nM ), a nd s lo p e = 1 .2 ± 0 .2 5 ; in N a C i (S ) ,p A2 = 7 . 4 3 ± 0 .0 5 (K,, 3 7

n M )an d s lo p e = 1 . 0 2 ± 0 . 0 4 .

tion dependency and w as not reproduc ibly de tec ted. To assess

w hether 1CI174 has neg ativ e ac tiv ity in intac t ce lls , therefore,

we r el i ed on th e m easu r em en t of i t s appar en tK ,. Concentra-

tion-response curves for the agonist DADLE in the presence of

increasing concentratio ns o f the negative antag onist w ere ex-

am ined (Fig . 9A ). S child analy sis o f these data (Fig . 9B ) y ields

an ap par en t K , value of 30 ± 7 nM , (three experiments ), w hich

is s im ilar to the value measured in membranes at high concen-

tratio ns o f N a’. This indicate s that no ligand-independent

ac tiv ity is demonstrable and that there is no s teady s tate

accumulatio n of RG complex es in the membrane of intac t ce lls .

1 0 2

1 0 1

0)

.3

1 0 0

L o g [IC I 1 7 4 ] M

F ig . 9 . A nt ag o n is m b y IC11 74 o f ag o n is t -m ed ia ted inhibition o f c AMP

a c c u m u la tio n in in ta c t N G 1 0 8 -1 5 c e lls . C o n flu e n t N G 1 0 8 -1 5 c e llser e

m e c h a n ic a lly h a rve s te d a n d re s u s p e n d e d in a s s a y b u ffe r (2 4 ) c o n ta in in g

1 0 0 M p h o s p h o d ie s te ra s e in h ib ito r R o 2 0 -1 7 2 4 a t 3 7 # {1 7 6 }n th e p re s e n c e

o r a b s e n c e o f s e le c te d c o n c e n tra tio n s o f DADLE a n d lC l1 7 4 , a s m

c a te d . In c u b a tio n la s te d 2 0 m m . Th e to ta l le ve l o f c AMP w a s d e te rm in e d

b y ra d io im m u n o a s s a y (2 4 ). A , R e p re s e n ta tive c o n c e n tra tio n -re s p o n s e

c u rve s fitte d b y a fo u r-p a ra m e te r lo g is tic m o d e lso lid lin es). D a ta w e re

n o rm a liz e d a s fra c tio n s o f th e le ve ls o f c yc lic n u c le o tid e m e a s u re d in t

a b s e n c e o f lig a n d . B , S c h iid p lo t o f th e d a ta . Inw o a d d itio n a l e xp e n -

m e n t s , K, va lu e s w e re m e a s u re d u s in g o n ly tw o c u rv e s , o n e in tp re s e n c e a n d o n e in th e a b s e n c e o f a n ta g o n is t. T h e m e a n va lu e a ve ra g e d

from th e th re e e xp e rim e n ts is g ive n in th e te x t.

D i s c u s s i o n

In this s tudy w e highlight three important features o

prote in-linked receptors. 1 ) A ligand’s efficacy , if de fined

molecular terms, can span a continuous spectrum of amplitudes

rang ing from positiv e to neg ative values. 2 ) M odulators (GT

sodium?) that reduce the stability constant M betw een receptor

and G pro tein can affect the sys tem in a manner that

inv erse ly re lated to the efficacy of the lig and. 3 ) Recepto

coupling in the absence of lig and w ill occur w hen the value

the stability constant M is not neg lig ibly small. These po ints

and the ir implications are discussed be low .

“ M olecu lar ef f i cacy ” and negat i ve an t agon i sm . B ecause

s ignal transduction at G prote in-linked receptors depends

the ability o f H to induce binding of R to G , the coupli

constant a, w hich measures amplitude and s ign of such

allos teric e ffec t, also defines the molecular eff icacy of the lig a

H. B ecause the log arithm of a can range betw een negative

pos itive values , ligands can display pos itive , neutral, or negative

eff icacy . S imulatio ns according to this concept predict features

that correspond very c lo se ly to experimental observatio ns.

The model predic ts that antagonists can inhibit G prote

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 10/12

5 5 8 C o s ta e t a! .

ac tiva tion accord in g to the d eg ree of nega tiv e e fficacy and be

partia l o r fu ll w ith respec t to the ex ten t o f inh ib ition , jus t as

ago n ists do w ith respec t to the ex ten t o f stim ula tion . A s w e

h av e sh ow n p rev io usly , o p io id an tag on ists can ex h ib it a w ho le

ran ge of rela tive m ax im a l effec ts fo r in h ib itio n of GT Pase , a s

op io id agon ists do fo r stim u la tion (14 , 1 5 ). 2 ) U nde r m ost

con d ition s, a lig an d w ith a 1 (neu tra l an tago n ist) w ill d isp lay

s im ila r p o tenc ies in com peting fo r ligands hav ing e ith er a > 1

or a < 1 . W e have show n prev ious ly tha t the benzom orp han

M R2266 , w h ich h as m in im a l e ffects on G TPase (i.e ., is a

pu tativ e neu tral an tag on ist) , d oes in deed com pe te fo r the in -

h ib ito ry ef fect o f the pep tide rg ic an tagon is t 1C 1174 (w hich

m axim ally in h ib its the en zym e) an d fo r the stim u la to ry e ffect

o f th e ago n ist (D ADLE ) w ith ve ry sim ilar pA 2 va lues (14 ) . 3 )

T he b in d ing iso the rm o f a n eg a tiv e an tag on is t (a < 1), u n lik e

tha t o f a neu tral an tago n ist, c an exh ib it app aren t he terog en eity

of site s, depend in g on the va lue o f a an d the presence of G .

W e have shown her e (F ig.7) tha t the b in d ing iso therm s of

the nega tiv e an tago n ist (e .g ., 1C 1174) a re in fac t cons isten t w ith

m ultip le a ffin ity s tates o f the recep to r, and th is he te ro gene ity ,

as d em onstra ted prev ious ly (14 ), d isappears up on in ac tiva tion

of the G p ro te in by pertussis tox in . It is im po rtan t to no te how

th is m od e l add resses som e fu ndam enta l q uestions o f theoretica l

pharm aco log y . 1 ) W hat is th e chem ica l na tu re o f a ffin ity an d

efficacy and w hat do these param ete rs te ll abou t recep to rs?

A ccord ing to the m ode l, a ffin ity is th e un co nd itio na l equ ilib -

r ium bind ing cons tan t (K ) fo r the b im o lecu la r in te rac tion of H

w ith R in th e ab sen ce o f G . E fficacy is , in stead , a ratio (re la ted

to the d iffe rence of free energ ies) be tw een the cond itiona l

a ffin ity cons tan t fo r the trim olecu la r in terac tion H R w ith G

an d th e a ff in ity K . Whe r e a s K dep en ds on ly on H and R and

is a u n ique property o f any ligand-recep to r pa ir,depends on

H , R , and G , so that an y l igand -r ecep tor pai r can have as m any

e ff icac ie s a s th e re a re ty pes o f G p ro tein s it in te racts w ith .

H ence, a ffin ity ch aracte r ize s recep to r b ind ing sites , b u t e ff icacy

a lso iden tifie s recep to r-G pro te in com p lex es , and the c lassifi-

ca tion of recep to rs acco rd in g to the true aff in itie s o f a lig an d

m ust inev itab ly be d iffe ren t from tha t based o n the spec trum

of efficac ies o f th e ligand . 2 ) C an affin ity and efficacy b e

d istingu ished an d independ en tly m easured? Y es , if pu rified R

and G are ava ilab le . A s elegan tly sh ow n by W eber (6 ) , the ha lf-

sa tu ra tion po in t fo r H bind in g to R in th e absence of G defines

1 /K. T he sh if t o f th is po in t in th e p resen ce of an ex cess o f G

y ie ld s a. F or the spec ial case of recep to r coup led to pertussis

tox in -sensitive G pro teins , w e pro pose a “non inv as ive” version

of th is experim en t; cells express ing a w ell iden tif ied recep to r

sub typ e cou ld be transfected w ith a vec to r co d ing fo r a k now n

a subun it o f th e G /G 0 fam ily of G pro te ins. If the leve l o f

expres sion can re su lt in a la rge s to ich iom etr ic excess o f G

pro te in w ith re sp ec t to recep to r, th en lig and b in d ing iso the rm s

be fo re and a fte r inactiv atio n by pe rtu ssis tox in can fu rn ish a

reasonab le approx im a tion of the tru e va lue o f. 3 ) C an aff in ity

an d efficacy be d isc r im ina ted in ph arm aco lo g ical s tu d ie s w h ere

on ly the concen tra tions o f H and /o r R are th e know n variab les?

N o , as p red ic ted b y the rec ip ro ca l na tu re o f the energe tic

lin kage be tw een the b ind ing p ro cesse s o f H and G th ro ugh R ;

the m o re the ligand H affec ts the b ind in g of G pro tein to the

recep to r , the g rea te r is the e ffec t o f G on the b ind ing of H .

T hus, any m ethod of ana lysis based o n the assum ption th a t

th e o bse rv ed o ccup ancy of the ligand is indepen den t o f its

e fficacy w ill re su lts in w ro ng estim a te s o f th e true aff in ity o f

the ligand . M ack ay (19 ) and K enak in and M o rgan (27 )

show n how the in te rac tio n of recep to r w ith G pro te ins inv

d a tes th e de te rm ina tion of agon is t a ffin ity based o n the ana lysi

o f ag on is t-m ed iated re sp onses in iso la ted tissu es. F o r rad io l

g and b ind in g s tu d ie s , w e sh ow here, as o the rs d id be fo re

17) , tha t the IC 50 va lu es o f various lig an ds depend on t

e ff icac ies an d on th e efficacy of th e trace r used to labe l R

3). W hen these sim ula ted da ta a re sub jected to ana lysis acco r

ing to the m ass-ac tion law , it is po ss ib le to de tec t up to t

apparen t b ind ing s ite s , ev en fo r a system sim ula ted fo r a s in

ty pe o f recep to r an d G pro te in (T ab le 1 ). I fw e fu rthe r p ostu late

the presence of tw o d ifferen t G pro teins , b o th in te rac tin g

R (27), i t is easy to im ag in e the case of a sing le recep to r t

can g en era te b ind ing and pha rm aco lo g ical da ta tha t a re

fec tly co nsis ten t w ith the ex istence of sev era l d istinc t

independ en t recep to r types. T he presen t s tu dy in trod uces

add itiona l reasons fo r skep tic ism . F irs t, the no tion th at an t

on is ts m ay also ex h ib it vast d iffe rences in e fficacy nega tes

comm on assum ption tha t the ir use in the ch arac te rization of

b ind in g site s af fo rds p ro tec tion from the in fluence of recep to r-

assoc ia ted G pro te ins. S econd , the find in g tha t the frac tion

preco up led recep to rs m ay be m uch h ig he r in iso la ted m e

bran es th an in w hole ce lls su ggests tha t, even fo r a s

recep to r-G pro te in p air, th e spec trum of ligand efficacies

i n vi tr o varies d ram atically w ith the degree of in tac tn ess o f

exp erim en ta l system . Thus the a ttem pt to d isting u ish recep to

he te rogene ity f rom transducer he te rogene ity u sing pharm aco-

log ica l s tu d ies and in th e absence of add itiona l m olecu la

know led ge is bo und to b e an unfru itfu l und ertak ing .

Stab i l i t y con stan t b et w een r ecep tor and G p r otei n

ef f ect of sod ium . For any rang e of v ar ia tion of th e stab ility

cons tan t M , th ere a re charac te ris tic re la tionsh ips b e tw een

ficacy a and changes in b ind ing propertie s and ac tiv ity o f

ligand . W e no ted prev iou sly (1 5 ) a p a radox ical d ifference

the w ay sod ium ions affect G TPase ac tiv ity an d ligand b ind in g

There w as an in ve rse re la tion sh ip be tw een the m agn itud e

the app aren t in trins ic activ ity (e ithe r n ega tive or pos itiv e)

the ligand fo r G TPase an d the ab ility o f sod ium to inh

GTPase in th e p re sence o f the lig an d , sugg esting tha t sod iu

ef fect is la rge r the c loser a is to 1 . H ow ever, d iffe ren t co nc

sions cou ld be draw n from the effec t o f so d ium on li

b ind ing . In th is case , N a inc reased the apparen t a ffin ity

an tag on ists tha t inh ib it G TPase , reduced tha t o f ag on ists ,

on ly neg lig ib ly af fec ted tha t o f partial agon is ts an d n eu t

an tag on ists . O ne m igh t take th is as stron g phen om eno log ica l

ev idence tha t the e ffec ts o f so d ium on ligand b ind in g and th

on lig an d-induced G TPase ac tiv ity a re tw o d is tin ct and u

la ted even ts . Ins tead , th is du al e ffec t o f sod ium can be

qua te ly exp la ined by a sing le m echan ism , if w e assum e tha t

ca tion selec tive ly reduces the m agn itude ofthe aff in ity cons tan t

M in the m em brane . U s in g a re la tive ly h igh va lue of(i.e ., 1 /

M nearly eq ua l to the recep to r concen tra tio n ) and the assum p

tion th at sod ium causes a 1 0-20-fo ld reduc tion of th is v

the m ode l can m im ic bo th the be ll-sh aped re lation be tw een

co ncen tra tion of sod ium and the in trin sic ac tiv ity o f the lig

fo r G TPase and the effec t o f sod ium on the b in d ing iso th erm s

o f ligan ds of d ifferen t eff icac ies. W e also prov ide ex perim en tal

ev id en ce , in iso la ted m em b ranes, tha t the appa ren t pA 2

pu ta tiv e nega tiv e an tag on ist, u n lik e tha t o f a neu tra l an tag

n is t, is in creased b y sod ium . T h is w as p red ic ted by sim ula tio ns

ind ica tin g tha t S ch ild p lo ts o f ligands w ith a < 1 , bu t n

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 11/12

Neg a t i v e Antagon ism 559

l i gands w i th a= 1, can be shi f ted to the lef t by reducti ons of

M .

By suggesti ng that the ef f ect of sodium acts l i ke a reduction

of M , w e d o n ot sp eci f y a p r eci se m olecu l ar m ech an i sm of

i nteracti on but simpl y manipulate an adm i ttedl y oversimpl i f i ed

model . Simulati ons only indicate that the phenomenology re-

lated to the introduction of sodium in the system is best

m im icked by a selecti ve reduction of the parameterM , rather

than changes ofK, a, or the rati o RGC. H ow ever , the ef f ect of

guanine nucleoti des on the binding of H can al so be modeled

by a reducti on ofM (7). A n “ M ef f ect” rul es out tr iv ial possi -

bi l i ti es, such as sodium competi ng w i th H for the same si te or

changing the chem ical properti es of H . B ut a host of indi sti n-

gui shable mechanisms remain possibl e, such as an acti on v ia G

( l i ke that of guanine nucleotides), a “ syntopic” competi ti on

w i th G , or a di rect ef f ect on R at a disti nct si te. B iochem ical

ev idence suggests that the latter i s the most probable (28).

T he net ef f ect of a modulator that reduces the stabi l i ty

constant M depends on i ts i ni t i al magni tude and thus on the

proporti on of receptor “ precoupl i ng.” This has tw o impl i ca-

t i ons. 1) I f a k now n receptor subtype i s expressed in cel l s

bearing di f f erent G proteins, qual i tati ve and quanti tati v e di f -

f erences in the ef f ect of N a (and, sim i l arl y , i n those of guaninenucleoti des) may serve to i ndicate di f f erences in the respecti ve

values of the stabi l i ty constant M . 2) H owever , because sodium

and sim i lar modulators may af fect the same receptor type

di f f erentl y in the presence of di f f erent G protei ns, any ef f ort to

cl assi f y unknow n receptor subtypes on the basi s of sodium or

GT P sensi ti v i ty is l i kel y to be as futi l e as the endeavor to

def i ne receptors on the basi s of the absolute potencies of

agoni sts i n di f f erent ti ssues.

R ecep t or act i v i t y i n t h e ab sen ce of l i gan d an d d i f f er -

en ces b et w een m em b r an es an d i solat ed cel l s. T h e m od el

predi cts that a di sti ncti on betw een neutral and negati ve antag-

oni sm is only possibl e i f the value of the af f i ni ty constant i s

l arge enough for the spontaneous reacti on betw een empty re-

ceptor and G protein to occur to a measurable ex tent. There-

fore, the demonstrati on of negati ve antagonism in a biological

response can al l ow one to determ ine w hether unoccupied re-

ceptor-G protein complexes accumulate as stable intermediates.

Thi s i s especial l y important f or the study of intact sy stems,

w here biological responses are separated f rom the ini ti al occu-

pation of the binding si te by a cascade of biochem ical steps

and, therefore, ‘ basal acti v i ty ’ i s no longer a di rect i ndi cati on

of the state of acti vati on of the G protein. W e show that l i gand-

independent receptor acti v i ty , w hich obv iously ex i sts in mem-

branes, does not occur i n the intact cel l . T he close sim i lari ty

betw een the pA 2 of 1C1174 obtained here in intact neuro-

blastoma cel ls and that measured prev iousl y i n i solated mouse

vas deferens (29) al so suggests that the negati ve antagoni sm of

thi s l i gand and, consequentl y , spontaneous receptor acti v i ty

are events only detectable in i solated membranes.

W hy do unbound receptor-G protein complexes accumulate

in the isolated membrane but not in the intact cel l ? A l i kel y

explanation i s that, i n the presence of cy tosol i c factors such as

G T P , i ons, and perhaps other as yet unknown regulators, the

interacti on betw een R and G is onl y transient and no longer

descr ibable by an equi l i bri um model (see also Ref . 19). This

means that, upon separati on of membranes f rom cy tosol , spon-

taneous receptor acti v i ty i s the inev i table consequence of an

interrupted balance betw een the intri nsic high af f i ni t i es of the

components present i n the membrane and the ex ternal

straints imposed by modulators ex isti ng in high concentrati ons

in the intact cel l . N egati ve ef f i cacy for antagoni sts has b

demonstrated at receptors that ei ther stimulate (30, 31)

i nhibi t (32) adeny late cycl ase. I n al l cases, isol ated membranes

or permeabi l i zed cel l s have been used. T hose data, therefore,

f ur ther corroborate the idea that an intact i ntracel l ul ar e

ronment i s a key factor i n prev enti ng spontaneous recepto

activ ity.

A re negati ve antagonists therapeuti cal l y useful and w or

searching for? N o, i f competi ti on w i th agoni st i s the m

concern. I n f act, af f i ni ti es being equal , the neutral antagoni st

i s a more ef f i ci ent agonist bl ocker than i s the negati ve k

H ow ever , onl y a negati ve antagoni st can suppress spontaneous

receptor acti v i ty . Recent f i ndi ngs indi cate that certai n mu

ti ons in G protei n-l i nked receptors resul t in consti tuti ve rece

tor acti v i ty (33). I f such mutati ons prove to occur in human

pathology , the search for and development of negati ve antago

ni sts is a novel important target of drug di scovery .

Acknowledgments

W e are grateful to D r . A ndrew Shenker f or cri ti cal reading of the manuscri pt.

References

1. Furchgott, R . F. T he use of -haloalky lam ines in the di f f erentiat ion

receptors and in the determ i nat ion of di ssociat ion constants of recept

agoni st com pl ex es. Adv. D r ug Res. 3:21 -55 (1966) .

2. B lack , J. D rugs f rom emasculated hormones: the principl e of syntopic anta

onism. Sci ence ( Washi ngton D.C . 2 4 5 : 4 8 6 - 4 9 3 (1989).

3. K enak in, T . P.T he P ha r ma co l og i ca l Analysis of D r ug-Receptor I nter action.

Raven Press, N ew Y ork , 31-51 (1987) .

4. L ef kow i tz, R . J., B . K . K obi l ka, J. L . Benov ic, M . Bov ier, S. Cotecchia,

P. H ausdorf f , H . G . D ohlman, J. W . Regan, and M . G . Caron. M olecula

biology of adrenergi c receptors. Coldpr ing H ar bor Symp. Quant. Bio l .

53:507-514 (1988).

5. G i lman, A . G . G proteins: transducers of receptor -generated signals.nnu.

Re v . Bi o c h e m. 56: 615-649 (1987).

6. W eber , G. Energeti cs of l igand binding to protei ns.dv. Pr otein Chem. 29:1-

83 ( 197 5) .

7 . DeL ean, A ., J. M . Stadel , and R. J. L ef kow i tz. A ternary complex m

explai ns the agonist-speci f i c binding properties of the adeny late cy clas

coupled f l -adrenergic receptor . J. B iol . Chem. 255:7108-7117 (1980).

8. W regget, K . A ., and A . D e L ean. The ternary compl ex model : i ts proper tiesand appl i cation to l i gand interactions w i th the D2-dopamine receptor of

anterior pi tui tary gl and. M ol . P har mocol . 26:214-227 (1984).

9. Burgisser , E., A . D e L ean, and R . J. L ef k ow i tz. Reciprocal modulation

agonist and antagonist bi ndi ng to muscari nic chol inergic receptor by guanine

nucleotide. Pr oc. Nat i . Acad. Sci . USA 79:1732-1736 (1982).

10. Y eung, S.-M . H ., and R. D . G reen. A gonist and antagonists af f i ni ti es

i nhibi tory adenosine receptors are reciprocal l y af f ected by 5’ -guany ly l im i -

dodi phosphate or N -ethy l mal ei mi de. J. Bi ol . Chem. 258:2334-2339 (1983).

11. Cerione, R. A ., J. Codina, J. L . Benov ic, R . J. L ef k ow i tz, L . B i rnbaumer,

M . G . Caron. The mammal ian f l -adrenergic receptor: reconsti tution of f u

t ional i nteractions betw een pure receptor and pure stimul atory nucleoti de

binding protein of the adenylate cy clase system.iochemistry 23:4519-4525

(1984).

12. Senogles, S. E ., J. L . B enov ic, N . A ml aiky , C. U nson, G. M i l l i gan, R. V i ni tsky ,

A . M . Spiegel , M . G. Caron. The D 2-dopam ine receptor i s functional l y

associated w i th a pertussis tox in-sensi ti v e G protein. J. B iol . C /ter n.

2 62 : 48 6 0 - 48 6 7 ( 19 8 7 ) .

13. L eung, E., and R. D . Green. D ensi ty gradient prof i les of A , adenosin

receptors label ed by agonist and antagonist radi ol igands before and a

d et er gen t so lu bi l izat io n. M ol Phar rnacol . 36: 412-419 (1989).

14. Costa, T ., and A . H erz. A ntagonists w i th negati ve intrinsic act i v i ty atelta

opioid receptors coupled to GTP-binding proteins.Proc. Nat! . Acad. Sci . U SA

8 6 : 73 2 1 - 73 2 5 ( 19 8 9 ) .

15. Costa, T ., J. L ang, C. Gless, and A . H erz. Spontaneous association betw

opioid receptors and GTP binding protei ns in nati ve membranes: regulati on

by antagonists and sodium ions.M o l. P hc ir rn ac ol . 37:383-394 (1990).

16. Gierschik , P., D . Sidi ropopulos, M . Steissl inger, and K . H . Jakobs. N a

regulati on of f ormy l pepti de receptor medi ated signal t ransducti on in H L

cel l s: ev idence that the cation prevents acti v ation of the G-protein by u

c up ied r ec ep to rs. Eur . J. Phar r nacol . 172:481-492 (1989).

17. Ehlert, F. J. T he relat ionshi p betw een muscari nic receptor occupancy an

adenylate cyclase inhibi tion in the rabbi t myocardium. M o! . P har macol .

28:410-421 (1985).

18. Ehlert, F. J., and B . E. R.athbun. Signal ing through the muscarinic recept

8/3/2019 Mol Pharmacol 1992 Costa 549 60

http://slidepdf.com/reader/full/mol-pharmacol-1992-costa-549-60 12/12

5 6 0 C o s ta e t a! .

ad eny late cyclase system o f th e he art is buf fered aga inst G TP ove r a rang e

ofconcen trat ions. M ol Phar macol . 38:148 -1 58 (19 90) .

19 . M ackay , D . In terp reta tio n o f re la tive po ten cie s, re la t ive eff ica cies and app ar-

en t a ffin ity cons tan ts ofagon ist d rug s es tim ated from concen tratio n- resp on se

curves. J. Theor . B iol . 142 :41 5-4 27 (19 90) .

2 0 . H iga sh ijim a, T ., K . M . Ferguson , M . D . Sm igel , and A . G . G ilm an . Th e ef fec t

o f GT P and M g on the G TP ase a ctiv ity an d the flu ore scen t prope rtie s of

G,, . J. B iOL Chern. 262:757-761 (1986) .

21 . Ro ss, E . M . S ig na l sor ting and am plifica tio n thro ugh G pro tein -co up led

receptors. Neu r o n 3:141-152 (1989).

22 . D e Lean , A ., P . J. M unso n , and D . Rodba rd . S im u lta neo us an aly sis of fam ilies

of sigm oidal cu rve s: ap p lication to b ioas say , rad io ligan d as say and physio lo g-ica l do se- respon se curves . Am. J. Physiol . 235:E 97-E 102 (1978).

23 . M unson , P . J. , and D . Rodb ard . L IGAND : a v ersatile app roa ch for the

ch arac ter izat ion o f ligan d-b ind ing system s. Anal . Biocher n. 107:220-239

( 1 9 8 0) .

24 . Cos ta , T . , M . W U ster , C . G ram sch , and A . H en . M u ltip le sta tes of the opio id

recep to r m ay m odu late ad eny late cy cla se in in tac t n euro b lastom a x g liom a

hyb rid cel ls. M o l. P ha rm o.c ol . 28:1 46-154 (1985).

25 . C o sta , T ., F .-J. K linz , L . V achon , an d A . H erz . O pio id rec ep tors are cou pled

tig h tly to G p ro tein s bu t loo sely to adenylate cyc lase in NG 108 -15 ce ll

membranes . M ol . P ha rma co l. 34:744-754 (1988) .

26 . V achon , L ., T . C osta , and A . H e rz. G TP ase an d adeny late cyclase d esensit ize

at d iffe ren t ra te s in NG 108-15 cells. o !. P har ma col . 31:1 59-168 (1987).

27 . K enak in , T . P .,an d P . H . M o rgan . Theore tic al ef fec ts of s ing le an d m ultip le

tra nsducer rec ep tor cou pling p ro tein s on e stim ates o f th e rela tive po ten cy

agonis ts . M ol P har macol . 35:214-222 (1989) .

28. H ors tm an , D . A ., S . B rand on, A . L . W ilso n , C . A . G uye r, E . J . C rago e,

L . E . L im b ird . A n a spa rta te conserv ed am ong G pro tein re cep tors con fe

allosteric reg ulation of a2-adrenerg ic receptors by sod ium .. B iol . Chem.

265:215 90-21 595 (19 90).

29 . M cKn igh t, A . T ., A . D . Co rbe tt, M . M a rco li, a nd H . W . K os terl itz . The opi

re cep tors in the ham s ter vas d efe ren s are o f theel ta -t yp e. N eu ro ph ar mo .co l-

ogy 24:1011-1017

30 . M urray , R ., and A . K . K een an . T he fl-a drenocep tor is precou pled to G

chicken e ry th ro cy te m em branes . C el l. Si gn al . 1 :17 3-1 79 (1 989).

3 1 . G #{246} tze , ., a nd K . H . Jakob s. E vid enc e for an ag on ist- free bu t an tago nis t-se nsi tive fl-a dreno cep tor ac tion in tu rkey ery thro cite m em branes .Naunyn-

Schmiedeberg’ s Ar ch. PharmacoL 341:R 30 (1 990).

32 . M a, H ., and R . D . G reen . M odula tion o f aden yly l cy cla se in de terg en t-

pe rm eabiliz ed chick ca rd iac m yo cite s b y A , adenosine recepto r agon ists

an tagonists. Bi ophys. J. 59:528 4 (1991).

3 3 . K jelsberg , M . A ., S . C otecch ia , J. O strow ski , M . C . C a ron , and R . J. L efk ow itz .

C ons titu tiv e ac tiva tio n of the a ,B -ad ren erg ic recep to r by all am ino

subs titu tio ns at a sin gle s ite.. BiO I . C/ter n. 267:1430-1433 (1992) .

S end repr in t req ues ts to : T omm aso Co sta , AB S , DCRT , N IH , B uild in g

9000 R ockville P ike, B eth esd a, M D 20892 .