mol pharmacol 1994 kuestner 246 55

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ABBREV IAT iONS : P C R, p oly me ra se c ha in re ac tio n; BH K, baby ham ste r k idney ; HEP ES , 4 -(2 -hyd roxye thy l)-1 -p ipe raz ineethanesu fton ic ac id ; EG TAe thy lene g ly co l b is (fl-am inoe thy l e the r)-N ,N ,N ,N - te traace tic ac id ; kb , k ilobase (s ).24 6

0026.895X/94/020248-10$03.00/0Copyrigh t C by The A merican S ocie ty for P ha rm aco logy and Exper im enta l T herapeuticsA ll rights of re p roduc tion in any fo rm rese rv ed .MOLECULAR P H A R M A C O L O G Y , 46 :246-255 . 1994.

C lon ing and C ha rac te riza tio n o f an Abundan t Sub type o f theHum an C a lc iton in R ecep to rROLF E . K UES TNER , R ACHEAL D . ELROD , FRANC IS J. G RANT , FREDER IC K S . H AGEN , JOSEPH L . KU IJPER ,S HANNON L . M ATTHEW ES , PATR IC K J. O H ARA , P AUL 0 . SHEPPARD , S TEVEN D . STROOP , D EBORAH L . THOM PSON ,THEODORE E . W H ITMORE , DAV ID M . F IND LAY , SOUHE IR HOUSSAM I, PATR ICK M . SEXTON , and EMM A E . M OOREZymoGen e t i c s In c ., S e a ttle , W as h ing ton 98 1 02 (R .E .K ., R .D .E ., F .J .G ., F .S .H ., J .L .K ., S .L .M ., P .J .O ., P .O .S ., S .O .S ., D .L .T ., T .E .W ., E .E .M .) ,ndS t. V in ce n ts Ins titu te o f M ed ica l R esea rch , F itz ro y , V ic to ria , 3 0 65u s tra lia (D .M .F ., S .H ., P .M .S .)R ece ived Janua ry 13 , 1 994 ; A ccep ted M ay 6 , 1994

SUMMARYW e have c loned and chara cte rize d a second fo rm o f the hum anca lc iton in recep to r from T47D ce lls . I t resem b les the c lo nee-scr ibed by G om et a !. [J . C lin . In ve st. 90:1 726 -1 735 (1992)]excep t tha t it la cks a 16 -am ino ac id in se rt in the pu ta tive firs tin tra ce llu la r lo o p . The in se rt-n ega tive recep to r a ppears to be th em os t abundan t fo rm , and it occu rs a t a re la tive ly constan t leve lin a ll exp re ss ing tissues . In con tras t, the inse rt-pos itive recep to ris fo und a t low le ve ls in m os t tissues bu t its exp ress ion le ve lsappea r to be m uch m o re va ria b le . The inse rt-n ega tive cDNA wass tab ly exp ressed in baby ham s te r k id ney ce lls . L ike th e endog -enous T47D recep to r, the recom b inan t re cep to r ha s an equa llyh igh a ffin ity fo r sa lm on and po rc ine ca lc iton in bu t a 3 -4 -fo ldlow er a ffin ity fo r h um an ca lc iton in . H igh concen tra t ions o f ca lc i-ton in gene -re la ted pep tide , ra t am y lin , s ecre tin , o r vasoac tive

in tes t ina l pep tid e do no t s ig n if ica n tly com pe te w ith ca lc iton in fob in d in g to the recom b inan t recep to r. CaJc iton in s t im u la te scAMP response in bo th T47D a nd tra ns fe cte d baby ham ste rk idne y ce lls . S a lm on ca lc iton in is m o re po ten t than hum an cac iton in fo r T47D ce lls , bu t thewo a re nea rly equ ipo ten t fo rhetrans fec tan ts . F urthe rm ore , th e ED fo r th e CAMP response int he t ra ns fe ct an ts is 1 0-1 00 -fo ld low er th an in T47D ce lls . a lc i -ton in s tim ula tes ino s ito l phospha te tu rnove r and e leva tes in te rna lca lc ium leve ls in th e tran sfe cta n ts . Th is re sponse requ ires non -phys io log ica l leve ls o f ca lc iton in and is d irec tly co rre la ted w iththe num be r o f re cep to rs . La stly , by us ing a hum an /roden t som a tic ce ll hyb rid pane l an d in s itu hyb rid iza tion , w e loca lize d thehum an ca lc iton in recep to r gene to ch rom osom e 7 .

C alc iton in is a pep tide ho rm one tha t is secre ted b y the Cce lls o f the thy ro id in re sp onse to a h ype rca lcem ic sig na l (1 ) .T he tw o m ajo r ta rg e ts o f calc iton in a re the k id ney , w he re its tim ulates the excre tion of ca lc ium and oth er e lec tro ly tes, andbon e , w he re it inh ib its bo ne re so rp tio n and hence ca lc iummob iliza tio n (1). D ue to its an tireso rp tive p roperties, ca lc iton inis u sed c lin ically fo r the trea tm en t o f a varie ty of b one d iso rders ,in clu d ing P age ts d isea se , hy pe rca lcem ia o f m a lign an cy , an dosteoporos is (1 ). T he ana lgesic e ffec t o f ca lc ito n in is w ell d oc-um ented , an d ca lc iton in recep to rs a re found in the bra in ,prim arily the hypo tha lam us . T he calc iton in recep to r is a lsoex pres sed in o th er tissues such as stom ach , p lacen ta, and lung ,bu t the p hysio log ica l ro le o f ca lc iton in in th ese tissues is n o tw e ll un de rs tood (1 ) .

T h e b io log ica l effec ts o fca lc ito n in a re th ough t to b e m ed iatedb y cAM P , an d pe rhaps by the ph osp ho lip ase C p athw ay (2 ) .T he refo re, it w as an tic ipa ted th at th e ca lc iton in recep to r w ou ldb e r ela te d to th e m u scarin ic an d adren erg ic recep to rs , w h ichh av e seven tran sm em b rane dom ain s and transm it th eir s ig na lsvi a e ffecto r G pro te in s (3 ) . H ow ev er , w hen the cD NA s encod in g

th e porc ine calc iton in recep to r an d the o pos sum para thy ro idhorm on e/p arathy ro id horm one-re lated pep tide recep to r w erec loned , it w as fo un d tha t n e ithe r h ad an y sign ifican t h om o logyto the c lass ica l sev en-transm em b rane dom ain recep to rs b u tthey d id have sig n ifican t hom olo gy to each o the r (4 , 5 ). H encthe clo n ing of these tw o recep to rs es tab lished th e ex istencea new cla ss o f G pro te in -co up led recep to rs . S ubseq uen tly , o threcep to rs h av e been c lon ed an d assign ed to th is fam ily . Th esein c lu de the recep to rs fo r secretin , g lu cag on , g lucagon -lik etide , g row th h orm on e-relea sing ho rm one , and vasoac tive in tetina l p ep tide (6 -9 ) .

R ecen tly , G o rn et a l. (10) repor ted the c lon ing o f a hum anca lc iton in recep to r cD NA from B IN -67 ova rian ca rc in om ace lls . T he pred ic ted am in o ac id sequ ence o f the hum an recep to ris 7 3% id en tica l to tha t o f th e prev iou sly c loned porc ine recto r , bu t th is h um an recep to r co n ta ins a 16-am ino ac id in se rtthe firs t in tracellu lar loo p tha t is no t fou nd in the porcsequence . Th e b io log ica l s ign ificance, if an y , o f th is in se rtno t ye t been de fin ed . Tw ofo rm s o f the ra t ca lc iton in recep to rhave b een c loned , w h ich d iffe r by the p resen ce o r absence


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T iC i

tiT 2

M l M2

C2

t2

C alc ito n in R ec e pto r s ub typ e s 24737-amino acid insert in the second extracellular domain ratherthan the first cytoplasmic loop (11, 12). The larger isoform isprimarily expressed in brain tissue, and it displays alteredbinding affinities forvarious calcitonins. A similar variant hasalso been described for the mouse calcitonin receptor (13).

In this communication, we describe the cloning and charac-terization of a second form of the human calcitonin receptorfrom T47D mammary carcinoma cells (14). T his receptor isvirtually identical to the human receptor described by Gornta t. (10), except that it does not contain the 16-amino acidinsert. W e compared the tissue distribution of the two receptorisoforms by using PCR. W e also prepared stable transfectantsof BHK cells, which express varying numbers of the insert-negative isoform of the human calcitonin receptor, and char-acterized them with respect to binding and second messengercoupling. T hese results are compared with the behavior of theendogenous receptor expressed by T47D cells.

Ma te ria ls a n d Me th o d sCloning of the receptor cDNA . RNA was isolated from T47D

cel ls usi ng guanidine isothiocyanate, followed by centrifugation in CsC1(15). Poly(A ) RNA was selected twice on an oligo(dT ) column (16).The Gubler-H offman method was used to generate doubl e-strandedcDNA , which was then ligated into the Zem228CC expression vector(8). I n this vector, the expression of the cloned cDNA is driven by amodif ied metallothionein promoter and the neomycin selection markeri s con t r ol led by th esimian virus 40 promote r.

The Primegen program (17) was used to identify regions that arehighly conserved between the porcine calcitonin, opossum parathyroidhormone, and rat secretin receptors. T hese sequences were then usedt o d esign low -degener acy ol igonu cleot i de PCR p r im er s. T heenseprimer was C l ( 5 - CA TCCACGGCAYAARAAYATGT TYY T - 3 ) andthe antisense primer wasC 2 ( 5 - A CTC TCCGGT TRCARAARCAR -TADAT -3 ). T he positions of these primers are shown in Fig. 1. U singthese primers, a 633-base pairragment, which was 80% identical tothe porcine calcitonin receptor, wasmplified from T47D cDNA . Thisfragment was isolated, radioactively labeled using a Stratagene Prime-I t kit, and used to probe the T47D cDNA library bytandard tech-niques. D N A sequenci ng was performed according to the method ofSanger et aL (18).

PC R analysis of various tissues. cDNA s that had been derivedfrom various tiseues known to express the calcitonin receptor werepurchased from Clontech. T he region containing the insert was ampli-fled f r om 1 ng of cDNA by using the sense primer ti (5 -TA CCCGCA -TACCAAGGAGAAG-3 ) and the antisense primer t2 (5 -A AGAGA -TAA TACCACCGCAAGC-3 ). O fthis reaction, 1/50th was reamplif iedusing t h e sense p r im er T i (5 -C CA TC AG AA AA GG TFA CA AA AT -3 )and the antisense primer T 2 (5 -CA CA GA GCA TC CA GA AA TA GT T-3 ). T he positions of these primers arehown in Fig. 1. A mplificationswere performed using Ampliwax PCR G em 5 (Per k in E lm er ) an d V en t

(exo) polyinerase (N ew England B iolabs). T he conditions for theirstamplification were 38 cycles of 45 sec at 94 45 sec at 58* , and 2at 7 2#{ 176} .he conditions for the second amplification were 20 cycles45 sec at 94* 45 sec at 58 and 2 mm at 72* . For both amplificationsa final extension wascarried out for one cycle at2 for 7 mm. ThePCR products from the second reaction were transferredto a H ybondN nylon membrane and hybridized w ith either (a) a 53-baseoligonucleotide that corresponds to a portion of the second transmem-brane domain and the f irst six amino acids of the first extracellularloop and should theref ore recognize both r eceptor i sof or m s or (b ) a 39-base pair oligonucleotide that corresponds exactlyo the insert foundbetw een posi ti ons 174 and 175 and should hybridize only w ith theinsert-positive form.

Insert-negative and -positive cD NA s were used as positive controls.T he in ser t -posi t i ve cD NA was generated as follows. Amplification wasperformed as described above. The region of the gel that correspondedin size to the product derived from an insert-positive cD NA wasurifiedand ligated into a pN EB193 vector. A fter transformation, positiclones were identified by their ability to hybridize to the insert-specificprobe. An EcoRI /Ns i I fragment containing the insert washen l igatedinto a linearized insert-negative cD NA , andts authenticity was con-firmed by sequencing. PCR amplification of varying amounts ofinsert-positive and -negative constructh demonstrated that neitherform underwent preferential amplification.

Cel l cu l t u r e and transfections. T 47D human mammary carci-noma cells (H BL 133; American Type Culture Collection) weretinely cultured in RPM I 1640 medium supplemented w ith 0.29 mg/mL-g lu tamine , 1 mM sodium pyruvate, 600 ng/ml insulin, 1M hyd r o-cortisone, and 10% heat-inactivated fetal calf serum. BHK -570 c(CRL 1632; American Type Culture Collection) were routinely growin Dulbeccos modified Eagles medium containing glutamine, pyruvate,and 5% heat-inactivated fetal calf serum. A ll cell lines were routinelypassaged with trypsin-ED TA and were periodically checked foryco-p ln .sma contamination using a Geneprobe kit.

BHK cells were transfected using a modification of the calciumphosphate pr eci pi tati on technique (19), and sta bl e tr ansf ec tan ts wereselected with 500 pg/ml G418 (GIBCO). Clones were isolated usingcloning rings and were maintained in the presence of G418.

Calcitonin binding. The cells were plated at0 cells/24-w ell platefor 48 hr before theassay. The cells were rinsed w ith binding medium(RPMI 1640 medium containing 1 mg/ml bacitracin and 1 mg/mlbovine ser um al bum in ) and in cu bated in t h isedium for 15 mm atroom tem perature. The medium w as then r em oved and r ep laced w i t hbinding medium containing a constant amount of ei t her 1n I ab eledsalmon calcitonin (800-2000 C i/mmol; Peninsula) or -labeled hu-man calcitonin (2000 C i/mmol; Amersham) and serial dilutionsunlabeled competitor. T he cells were incubated for 1.5 hr at rtemperature, washed three times with phosphate-buffered saline,ndsolubilized w ith 0.25- i N NaOH . T he sam p les w er e col lected f r om eachwell and counted on a Cobra-auto y cou nt er . Scatchard analysis wasperformed using the K inetic, EBDA L IGAND , Lowry programromBiosoft (20).

F ig . 1 . S ch e rn a c d ia g ra m th a t s h o w s th e lo c a -tio n o f va rio u s P C R p rim e rs . Ha tched boxes,s e v e n t r a n s m e m b r a n e d o m a i n s o f th e re c e p to r.P r im e r s C l a n d C 2 w e re u s e d to a m p lify a 6 33 -b a s e p a ir fra gm e n t o f th e c D NA, w h h w a s th e nu s e d to p ro b e th e C O NA lib ra ry . A s e t o f n e s te dp rim e rs , tl/t2 a n d T1 /T 2 , fla n k th e 1- am i n oa c id in s e rt in th e firs t c y to p la s m ic lo o p o f th eh u m a n c a lc ito n in re c e p to r . T h e s e w e re u s e d toe v a l u a t e th e re la tiv e f r e q u e n c y o f th e re ce pto r

O O H s u b typ e s in v a rio u s tis s u e s . P rim e rs M1 /M2w e re u s e d to e ntify th e c h ro m o s om a l io c a o no f th e h u m a n c a lc fto n in re ce p to r g e ne . insert A ,l o ca t i o n o f th e 1 6 -a m in o a cid in s e r t in th e h u m a nr e c e p t o r ; insert B, l o ca t i o n o f th e 3 7 -a m in o a cidin s e rt fo u n d in th e m ou s e /ra t c a lc ito n in re ce pto r.


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248 Kuestne r e t a !.

R . E . K uestner, and E . E . M oore , unpu b lished observ atio ns.

cA MP m ea su rem en ts . T o m ea su r e cAM P r e sp o n se, 1 -3 iO cellsw ere p lated in 2 4 -w ell p late s f o r 1 -2 day s b ef o re th e assay . C e lls w ereex posed to v ary ing concen tratio ns o f hum an or salm on calc iton in , inse rum -co n tain in g m ed ium w ith 10M isobu ty lm e thy lx an th in e, f o r 1 0m m at 37 Forsk o lin (25 M ) w as in clu ded as a po sit iv e con tro l. T hereac tion w as stop ped by the ad d itio n o f 4 v o lum es o f b o ilinga t e r .S am p les w ere then e ither f ro z en o r assay ed im m ed iate ly u sing anA m ersham S c in tillation Prox im ity cA M P assay sy stem . A ll po in ts w ereassay ed in trip licate.

Inos ito l pho sphate m easurem ent. C ells2 x iO w ere p lated ina 24 -w e ll p late f o r 24 hr and then labe led ov ern ig h t w ithzC i /ml myo-[2 -3H jin osito l (sp ecif ic activ ity , 20 C i/m m ol; A m ersham ) in inos ito l-free Dulbeccos m od if ied E ag le s m ed ium con tain ing g lu tam in e, py ru -v ate , and 10% dialy z ed serum . T he ce lls w ere th en w ashed w ith m ed iumbu f f e red w ith 20 m M H EPES , pH 7 .0 , con tain ing 10 m M lith iumch loride . T he w ash m ed ium w asep laced b y the sam e m edium contain-ing increas in g concen tratio ns o f hum an o r salm on calc ito n in . C ellsw ere incu bated f o r 30 m m at 3T . T he reac tion w as te rm inated by theadd ition o f co ld D u lbecco s m od if ied E ag le s m ed ium /10% perch lo ricacid (1:1). ED T A w as added to th e ly sate s to a f in al concentratio n o f 2m M , and the sam p les w ere neu traliz ed w ith.5 M K OH in 60 m MHEPES buf f er. T he labe led in osito l p ho sphates w ere iso lated on anA m ersham A M PR EP co lum n accord ing to the m anuf acture rs in stru c-t i o n s and w ere co un ted in a B eck m an 51 800 sc in tillation cou n te r. A llpo in ts w ere assay ed in trip licate .

C alc ium analy sis. T he quan titation o f in te rnal calcium lev e ls w asperform ed as d esc rib ed p rev ious ly (21 ). T he ce lls w ere p lated at adens ity of 3 x i0 ce lls/m l in uncoated , co v erg lass -w ell, tw o-cham bers lide s (N unc) and w ere incubated fo r 48 hr. T he s lide s w ere then rin sedw it h im a g in g b u ffe r (14 0m M N aC l, 10 m M glucose , 5 m M K C 1, 0 .5 -1 .8m M C aC12 , 0 .5 m M MgC1 2 , 10 m M HEPES , pH 7 .4) . T h e cells w ereloaded w ith f u ra-2 /ace to x y m e thy l e s te r (10 g /m l in im ag ing bu f f e r)f o r 30 m m at room tem peratu re and then rin sed th ree tim es. S lidesw ere m oun ted on a N ik o n E p ipho t inv erted m icro scope , and the ratioof fu r a -2 em iss ion a t exci t a t ion w a velen g th s o f 3 40 a n d 380 nm w a sreco rd ed ev ery 5 sec.T he data w ere analy z ed on a S u n com pu te r usingan Inov is ion so f tw are sy s tem design ed f o r ratio im ag ing . Fura-2 3 40 :380ratios (R ) w ere con v erted to calc ium concen trations , af te r calib ratio n ,u sing the f o llow ing f o rm u la: calc ium ( nM ) = 2240 [( R - 0.3) /(20 -R)]. T y p ical basal v alues o f in te rnal calc ium lev e ls ranged f rom 80 to150 nM .

Chrom osom e m app ing . T he H um an G en etic M u tan t C e ll R epos -ito ry H um an /R oden t S om atic C e ll H y b rid Pane l 2 (N ation al In s titu teof G eneral M edical S cience s, C onch Institu te f o r M ed ical R esearch,Cam den , N J) w as used in PCR am p lif ication to iden tif y the som atichy brid that con tain s the hum an calc ito n in recep to r g en e . T he senseprim er, M l, corre spo nds to a 21 -base pair s tretch o f an ad jacen t in tron(5 -GG T T GGG T A TG A CT GGT G T A G -3 ). T h is in tron sequ ence w asob tained f rom a 6 .5 -k b H ind III f ragm en t o f the calc iton in recep to rgen e t h a t h a d been id en tif ied by its ab ility to hy brid iz e to the cD N Ainsert. T he location s o f b o th the sen se prim er and the an tisense prim er,M 2 (5 -C T G G G C AG AAC T G AT AG G AC A -3 ), a r e sh ow n in F ig . 1 .T he PCR am plif ication w as perf o rm ed w ith00 ng o f each DN Asam p le . T he cond itions w ere 3 0 cy c le s o f 1 m m at 94 1 m m at 62 an d 1 m m at 72 T h is w as follow ed by a-m m f inal ex tens ion at 72* .T he pred ic ted p roduc t is 198 b ase pairs.

I n situ hybr id iza t ion wa s p er for m ed on h um a n m e t a p h a se ch r om o-som e sp r e a d s , w h ich w er e p r ep a r ed fr om p r im a r y h um a n lu n g ce lls a sdescribed prev ious ly (22) . A genom ic probe fo r the hum an calcitoninrecep to r, DM PC -H FF#1-130 1 (11D ) (se rie s A ), w as o b tain ed b y screen -ing a hum an P1 lib rary (G enom e S y s tem s, Inc .). A cen trom eric p robesp ecif ic f or chrom osom e 7 (D 7Z 1) w as ob tained f rom O ncor. T h e prob esw ere labe led by n ick -trans lation w ith b io tin -11 -dU T P (S igm a). T h euninco rpo rated nu cleotides w ere rem ov ed us ing a M ini-S pin G -50co lum n (W orth ing to n ), and the siz e o f the labe led probeas ver i f ied

on a 0 .7% agaro se gel .T he h y brid iz atio n w as perf o rm ed as describ edp rev iou sly (23 ), ex cep t th athy brid iz ation w ith th e calc iton in recep to rg en e p r ob e w a s carried ou t in 50% (v /v ) f o rm am ide , 10% d ex transulfate, 30 0 m M N aC 1 , and 30 m M N ac itrate (2xtandard saline citrate)at 37* for 6 h r be fore the addit ion o f the centrom eric p rob e. A tp o in t the f o rm am ide con cen tration w as raised to 65% (v /v ), andhy brid iz ation w as con tinued ov ern igh t. Prep arations w ere v iew edp ho tograp hed using an O ly m pus B H -2 m icroscop e w ith a B H -2 R FCflu o r escen ce a t t a ch m en t a n d E k t a ch r om e 400 f ilm .

Resu l t sC lo n in g of a n a lt e r n a t iv e fo r m of th e h u m a n ca lcit on in

recep tor. A n ex pression cDN A lib rary w as prepared fT 4 7D ce lls , a hum an m am m ary carcinom a ce ll line k now nex press the calc iton in recep to r (i4 ) . A 633 -b ase pair f ragm en to f the hum an calciton in recep to r w as am p lif ied f rom th is cDNb y PCR using degenerate prim ers th at co rrespond to reg ionthat are h igh ly co nserv ed be tw een th e porc ine calciton in , opsu m parathy ro id horm one , an d rat secre tin recep to rs. Tf ragm en t w as then u sed to probe the cDN A lib rary . A sinf u ll- leng th c lone w as f oun d am on g 40 0 ,000 c lones . T h is c loco n tain s a 3 .3 -k b in sert, o f w h ich 1 422 base pairs co nstitu teo pen read ing f ram e. T here is a long 3 un tran slated reg(1 871 base pairs) and a short 5 sequ en ce (3 7ase pairs) thatdoes no t ex tend to the p o in t w h ere G ornt al. (10 ) f ound asecond in -f ram e A UG start site . T h e cod ing sequence encod ea 47 4 -am ino ac id pro te in th at is 70% iden tical to th e porc icalc iton in recep to r sequence and is v irtually iden tical toh um an calc ito n in recep to r cD N A iso lated by G ornt al. (10 ) ,w ith the f o llow ing ex cep tions . M o st no tab ly , our c lone doescon tain an in sert o f 16 am in o ac id s be tw een pos itio ns 1 7417 5 and in th is regard is m ore sim ilar to the porcine recep to rT he absen ce o f th is in se rt in our c lone ind icates that attw o sub ty pes o f the h um an calc iton in recep to r ex ist. In ation , th ere is a sing le nu cleo tide d if f eren ce at pos ition 1w hich resu lts in the substitu tio n o f leuc ine fo r pro line inc lone . T h is d if f e rence m ay re f lec t in d iv idual v ariation insou rce o f the c loned D N A , or it m ay rep resen t a c lon ing artif ac tFig . 2 show s an alignm en t o f ou r c lo ne w ith o ther k nohom o log ues o f th is recep to r sub f am ily .

T is su e d ist r ib u t ion of r ecep t o r su b t yp es. O u r r esuin d icate that at leas t tw o alte rnativ e f o rm s o f th e humcalciton in recep to r ex is t. W e u sed a PCR app roach to de te rm inew he ther th ey are ex pressed in a tis sue -spec if ic f ash io n .thoug h th is approach is n o t stric tly q uan titativ e , w e dem ostrated that the in sert-pos itiv e and -neg ativ e recep to r con -struc ts are am p lif ied equally w e ll un der our PCR cond itions(data not show n). T here f ore , it s eem ed reasonable to useapproach to assess th e re lativ e d istribu tio n o f th ese tw o stypes in a v ariety o f tissues. W e o b tained cD N A s d eriv ed f roa v ariety o f tis su es k now n to ex press th e calciton in recep to rT hese inc luded k idney , hy p o thalam u s, s tom ach , f e tal b rain ,ce rebral cortex , mammar y gland , cerebe llum , p lacen ta, ov ary ,lu ng , and bon e m arrow . PCR prim ers that f lank the reg io nthe in sert con tain ing 16 am in o ac id s w ere used to am p lif yreg ion o f the calciton in recep to r f rom each o f th e cD N A sourcesA s a po sitiv e con tro l, w e also am p lif ied cDN A s represen tin gbo th th e in sert-pos itiv e an d -negativ e f o rm s o f the recep to rT he PCR products w ere separated on a 2 .5% agarosetrans f e rred to a ny lo n m em brane , and hy b rid iz ed either w ithpro be that shou ld in te rac t w ith bo th f o rm s o f the recep to rw ith an o ligon uc leo tide that sh ou ld hy brid iz e o n ly to the in ser


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a aHU CTR NRF T F TSRCLALF LLLNHP TP I L P AF S - NQTYP TI E P K PF LYWGRKKMMDAQYKCYDRN - QQLPAYQGEGPYCNRTPORCTR NRF TLTRWCLTLFI F LNRP L P VLP DSADGAHTP TLE PE P F LY I LGKQRMLEAQHRCYDRM - QKLPPYQGEGLYCNRTHUPTHR MGTARI APGLALLLCCPVLS S GYA LVDADDVMTKEEQI FLLHRAQAQCEKRUCEVLQRPASI MESDKGWTSAS TSGKPRKDKASGKLYPESEEDKEAPTGSRYRGRPCLPE 1OPPTHR NGAPRI SHSLALLLCCS VLS S VYA LVDADDVI TKEEQI l LLRNAQAQCEQRLKEVLR- VPELAES AKDSRSAKTK- - KEKPAEKLYPQAEESREVSDRSRLGFCLPE 1RAP THR NGAARI AP SLALLLCCPVLS SAYA LVDADDVFTKEEQI FLLHRAQAQc DKLLKEVLNTMNI MES DKGWTPAS TS GKPI KEKAS GKF YP ES KEP 4KDVPTGS RRRGRP CLPE 1RAGLPR NAVTPS LLRLALLLLGAVG- - RAGPRPQGATVS LS ETVQKUREYRI 4 QCQRF LTEAPLLATGLFCNRTRASECR NLS TMRPRLSLLLLRLLLLTKA AHTVGVPPRLDVRRVLLEERAHCLOQL S KEKKGALGPETASGCEGLRAVI PR MRPPS PPHVRWLCVLAGALACALRPAGSQA AS PQHECEYLQLI EI QRQQCLE EAQLENETTGCSKMHU GLR MPPCQPQRPLLLLLLLLACQPQVPS A QVNDFLFEKW CLYGDQC$ H NLSLLPPPTELVCNRTRAGLR NLLTQLHCPYLLLLLWLSCLPKAPS A QVNDFLFEKW CLYS DQCHH NLSLLPPPTELVCNRTHUGRFR MDRRI 4 WGAHVFCVLS PLPTVLG HNHPECDFI TQLREDESACLQ AAEENPNTTLGCPATRAGRFR MDSLLWAI %J VLCLLNLWGVALG HLHLEc DFI TQLRDDELACLQ AAEGTNNS SMGCPGTNUGRFR MDGLMWATRI LCLLS LCGVTLG HLHLEc DFI TQLRDDELACLQ AAEGTNNTS LGCPGT

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a a# # # # 1 a t 4 # u 1 1 1 apH#va#iaai iaua####4 mamai i i u au u iu2. 3. 4.HU_ CI R 4PORCI R W LSVDTNLLYI I HGPVMAALWNFFFLLNI LRVLv KKLKES O- - - EAES HMYLKAVRATLI LVPLLGVQFWLPWRPS TPLLG- - - KI YDYWHS LI HFQGFFVAI I YCFCNHEVQGALKRQ 4HUPTHR I LLS SG- NKKW 4OPPTHR b LS SG- NKkW I QVP I LUI WNFI LF I P4 I I RVLATKLRETNAGRCDTRQQYRKLLKSTLVLMPLFGVHYI VFMATPYTEVS GI LWQVOMHYEMLFNSFQGFFVAI I YCFCNGEVQAEI KKS 4RAPTHR l LS SG- HKKW I QVPI LASWLNFI LF I P4 I I RVLATKLRETNAGRc DTRQQYRKLLRSTLVLVPLFGVHYTVFMALPYTEVS GTL QMHYE$ LFNS FQGFFVAI I YCFCNGEVQAEI RKS 4RAGLPR 4RASECR NANASVl i JVI RGPVI LS l LI NF I FFI NI LRI LMRKLRTQETRGSETNHYK- RLAKS TLLLI PLFGI HYI VFAFS PEDAM EVOLFFELALGSFQGLWAVLYCFLWGEVQLEVQKK 4RAVI PR 4HU_ GLR 4RAGLR 4HUGRFR 3RAGRFR WDLDDS S PYWW I KGP I VLSVGVNFGLFLN1 I CI LLRKLGPAQGGLI I TRAOYW RLS KS TLLLI PLFGI HYI I FNFLPDS AGLG- - - - I RLPLELGLGS FQGFWAVLYCFLWQEVRTEI SRK 3NUGRFR I KLDNS S PCW I KGP I VLS VGVNFGLFLNI I CI LLRKLEPAQGGLHTRAQYW RLSKSTLLLI PLFGI HYI I FNFLPDS AGLD- - - - I RVPLELGLGS FQGF I VAVLYCFLNOEVRTEI SRK 3

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HUCTR WAQFKI QWWORU- GRRPSNRSARAAAAAA EAGDI PI YI CHQELRP4 EPAPJ NOGEES AEI I PLNI I EOES S A 4PORCTR WNQYO- - - AQRWAGRR- S TRAANAAAATAAAAAALAETVE I PVYI CHQEPREEPAGEEPWEVEGVEVI AMEVLEQETS A 4HUPTHR WSRWTLALDFKRKARSGSSSYSYGPMVSHTSVTNVGPRVGLGLPLSPRLLPTATT - - NGHPQLPGHAKPGTPALETLETTPPAMAAPKDDGFLNGS CSGLDEEAS GPERPPALLQEEWEI VN 5OPPTHR WSRWTLALDFKRKARS GS STYS YGPMVS HTS VTWVGPRGGLALSLS PRLAPGAGAS ANGHHQLPGYVKHGS I SENSLPS SGPE- PGTKDDGYLP4 GS - - GLYEPMVG- EQPPPLLEEERETVP4 5RAPTHR WS RWTLALDFKRKARS GS S SYSYGPHVSHTS VTNVGPRAGLS LPLS PRL- PPATT- - NGI I SQLPGHAKPGAPATETETL- PVTP1 AVPKDDGFLNGSCSGLDEEAS GS ARPPPLLQEEWETVN 5RAGLPR WERWRLERLNI QRDS SMKPLK- - CPTS S VS SGATVGS SVYAATCQNS CS 4RAS ECR WRQWHL- - - QEFPLRPVAFNNSFSNATP4 GPTHS TKAS TEQS RS I PRAS I I 4RAVI PR WRRWHLOGVLGWS SKSQHPWG- GS P 4 GATCSTQVSMLTRV- S PS ARRS S S FQAEVSLV 4HU_GLR WHRWRLGKVL -WEERNTSNHRAS S S P G - - - HGPPSKELQFGRGGGSQ- - - - DS S AETPLAGGLPRLAES PF 4RA_ GLR WRRWOEGKAL - QEERMAS S HGS HMAPAGTCHGDPCEKLQLMSAGS S S GTGCEPSAKTSLAS S LPRLADSPT 4HUGRFR WHGHDPELLPAWRTRAKWTTPSRS AAKVLTSMC 4RAGRFR WYGHDPELLPARRTCTEWTTPPRS RVKVLTS EC 4NUGRFR I J YGI 4 DPELLPARRTCTEWTTPPRSRLKVLTSEC 4Fig . 2. Alignment of human calcitonin receptor ( H U . C T R ) , porcine ca la tonin re ce ptor ( P O R C T R ) , human, opossum , and ra tpa ra thyroid horm onereceptors (H U P T H R , O P P T H R , an d R A P T H R , respective ly), ra t glucagon-like peptide 1 receptorR A G L P R ) , ra t se cre tin re ce ptor ( R A S E C R ) , ra tva soa ctive inte stina l pe ptide re ceptor ( R 4 W P R ) , human and ra t gluca gon re ce ptors ( H L L G L R and RA...G LR , respective ly), and human, ra t, andmouse growth hormone -re le a sing hormone receptors (H U G R F R , P A G R F R , an d M U G R F R , respective ly). T he puta tive transmembrane doma ins a reu n d e r s c o re d (# # # ) an d n u m b e r e d . * Cysteine residues in the extrace llula r doma ins. All prote ins a reu m b e re d (rig h t m a rg in ) fro m the initiatingmethionine. Location of the inse rtion in the ova iian ca rcinoma cDNA tha torresponds to the loca tion of an intron in the human g lu ca go n re ce pto rgene . T his ve rsion of the humancalcitonin receptor sequence has been deposited inenBank and EM BL sequence da tabanks (accession numberX69920 ) .


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25 0 Kuestne r o f a !.

A I 2 3 4 5 6 7 8 9 1 0 1 1 - +

B I 2 3 4 5 8 7 8 9 1 0 i i - +

Fig . 3. cONAs O b ta ined from vanous tissues w e re PCR a rnp litle d us ingpnm ers tha t flank the reg ion con ta in ing the in se rt. CO n tro lS in c luded bo thinsert-negat ive (-) and -pos itiv e (+ ) cDNA . A , S outhe rn b lo t h yb r id izedw ith a p robe th at re co gn iz es bo th fo rm s o f the recep to r; B , S ou the rnb lo t h yb rid ized w ith a p robe tha t is spec ific fo r the in se rt.a n e 1 ,s tomach ; ! an e 2 , l u ng ; !a n e 3 , ovary ; lane 4 , k id ne y; lane 5 , bone m arrow ;la n e 6 , m am mary g land ; la ne 7 , w ho le fe ta l b ra in ; la n e 8 , hypo tha lamus ;la n e 9 , ce rebe l l um ; la ne 1 0 , c ere bra l co rte x; la n e 1 1, p lacen ta . T hean tic ipa ted s ize fo r the p roduc t am p lifie d from the in se rt-nega tive formis 408 base pa irs and tha t fo r the p roduc t am p lifie d from the in se rt-pos itive fo rm is 456 base pa irs .posi tiv e f orm (Fig. 3). T he insert-negati ve f orm of the receptorw as expressed at a f ai rl y constant l evel i n al l ti ssues exam inedand appeared to be the most abundant f orm of the receptor. I ncontrast, the expression of the insert-posi ti ve receptor w asapparentl y much more variable. I t w as f ound at low levels inmost tissues, but i ts l ev el of expressi on ranged f rom beingundetectable in stomach, fetal brain, and cerebral cortex toconsti tuti ng almost hal f of the transcri pts i n ovary and pla-centa.

I nterestingly , these PCR primers should al so ampl i f y theregion that encodes the second ex tracel l ular domain, w here a37-amino acid insert has been f ound in the rat and mousecalci tonin receptors (1 1-13). I f an equivalent i soform ex ists f orthe human receptor, the predicted si ze of the PCR product i s519 base pai rs. Faint bands in thi s region of the gel w ereobserv ed for k i dney , mammary gland, hypothalamus, and pla-centa, and thi s may indi cate that thi s subtype is expressed atlow levels by these ti ssues. I n contrast to the rodent system,we did not observe a pref erenti al high level of expression ofth is subtype in any of our brai n samples. H owever, in f etalbrain a PCR product w as produced that i s i ntermediate in si ze,compared w i th the insert-posi ti ve and -negati v e f orms. A ddi -ti onal studies are underw ay to characteri ze thi s product and toveri f y that a human cal ci tonin receptor subtype ex i sts that i sanalogous to the rat brain i soform .

B inding. To assess the biologi cal acti v i ty of the insert-negati v e form of the receptor, it s cDNA w as transf ected intoB HK cel l s, and mul ti ple stable transfectants w ere i solated.Tw o of these, H x i and Hx2, w ere character i zed in detai l , andthei r behav ior w as compared w i th that of T47D cel l s. T herecombinant receptor expressed by both transfectants bound 9- labeled salmon cal ci toni n i n a speci f ic f ashi on; the l evel ofnonspeci f i c bi ndi ng w as


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5)E0.U

.01 .1 1 1 0 100 1000 10000

Competitor ( n M )

0.3

0.1

100B o u n d (f m o le s )

Calciton in R ecep to r sub ty pes 251

Fig. 4 . C om pe tition with binding of a l-Ia -sC T ba led sa lmon ca lcitonin to H xi ce lls by in-creasing concentra tions of unlabe led sa lm onrAmylln ca lcitonin (scfl, porcine ca lcitoninpOT) , hu-

man caictionin (hCT) , ra t a mylin (rAmylin),human ca lcitonin gene -re la ted peptide( hCGRP) , va soa ctive inte stina l pe ptide (VIP) ,and human secre tin (hsecretin). Maximumbinding in the absence of compe titor rangedfrom 22 ,000 to 27 ,000 cpm /we ll.

hCGRPVI PhSecre t inp CTh CT

w0)I -U -C0

Fig. 5 . Sca tcha rd ana lysis of binding of humancalcitonin to Hxi ce lls,pe rform ed a s de scribe d in M ate ria ls and Methods.TABLE IB in din g c ha ra ct o# {2 41 }s tl cs of T 47D , Hxl, and H x2 ce lls

1470 Hx 2 Hx lR eceptors/ce ll 7 -40 ,000 iO O ,000 800,000Kd

Humancalcitonin 2 . 1 0 . 3 1 .70 .1 42 .5Sa lmon ca lcitonin 0 .6 0 .5 0 .3 0 .01 0 .5 0. 2

EG T A , a calcium chelator , dem onst r at ed t hat t he p r im ar yresp on se is du e to th e m ob ilizatio n o f in te rn al ca lc ium sto re s .H ow ever , t he decay of t he r esponse w as m or e r ap id in theabsence o f ex te rna l ca lc ium , su ggestin g th at a secon d com po -nen t o f the re spon se m ay b e m ed iated by a ca lcium ch an ne l.

A lthough ca lc ium respon ses w ere a lsoee n in H x2 transfec-tan ts and T 47D ce lls , o n ly 30-60% of th e H x2 and 10 -2 0% ofthe T 47D cells show ed a re spo nse . A sim ila r reduc tion inam plitude w as seen .

Inosi to l phospha te produ ctio n . T o de te rm in e w h e th e r thein c rea sed m ob iliza tion o f in terna l calc ium sto res is m ed ia tedby inosito l trip hosp ha te , w e ex am ined the e ffect o f ca lcito n ino n in osito l ph osp ha te tu rno ve r. W e found th at ca lc iton ind uced a sign if ican t in crease in inos ito l ph osph a te tu rnov erH xl cel l s (F ig. 8). F or k sol i n (25sM ) d id no t indu ce th isresponse , w hich ind ica tes tha t it is no t an ind irec t con sequenceo fe leva ted cAM P lev els (da ta no t sh ow n). T ab le 3 is a summ aryo f the ED se v a lues, w h ich w ere ave raged from seve ral expe ri-m en ts . A s obse rved fo r the cAM P resp onse, the ED se va lu esw ere iden tical fo r sa lm on and hum an calc iton in . H ow ev er ,app ro x im a te ly 100 -fo ld h ighe r lev els o f ca lc iton in w ere requ iredto tr igge r an in osito l p hospha te re sp onse, com pared w ithcA M P r esponse.

Paralle ling the re su lts w ith the ca lc ium s tud ie s , w e fo un2 0 0 tha t the ino sito l pho spha te resp onse in H x2 ce lls w as

than tha t in Hx l ce lls and tha t no resp onse cou ld be d etec tedin T47D ce lls . T he ino sito l p ho spha te a ssay is p robab lysensitive enough to de tec t the low er percen tag e o f T 47D cth at sh ow a ca lc ium respon se w h en the ce lls a re m on ito reda sing le -ce ll lev el.

O ur resu lts sug gest th at th e ino sito l ph osph ate re sp onse m ao ccur o n ly in ce lls th at ex p re ss a su ff icien tly la rge num berrecep to rs . W e the re fo re ch aracte r ized seve ral o the r BHK tran s-fec tan ts , w hich exp re ss d iffe ren t leve ls o f ca lcito n in recep to rs ,fo r th eir in osito l ph osph ate re spon se . F ig . 9 show s th at, in thseries o f transfec tan ts , the m agn itu de o f th e inos ito l pho sph atere sp onse ap pears to b e a d irec t func tion of the n um berrecep to rs .

W e also at t em p t ed t o ob t ain T 47D t r ansfectant s that w ou ldexp r ess a lar ger n um ber of r ecom b in an t r ecep t or s, t o d eter m inewhe the r the re w ou ld be an accom pany ing inc rea se in th eirino sito l ph osph a te re sp onse. U nfo rtu na tely , the ex press ion 1ee ls o f the transfec ted recep to r in T 47D cells w ere tooow foru s to m ake th is as ses sm en t.

M app ing. T o id en tify the chrom osom al loca tion of thehum an cal ci t on i n r ecep tor , w e used CR pr im er s cor respond ing


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A

0 0.001 0.01 0.05 0.1 0.5s CT (n m )

500

340

200EUCs 70C)

Cs

= 5005)C.)CSC

- 340

200

70

1 10

20 n M sCT

100 200S e c o n d s

300

B

400

252 Kuestner ef aLA

9080

- 70 605o40 30I.- 20

100

B70000

- 600000) 50000

, 40000. 30000

l 2000010000

01 3.2 10

F ig . 6 . Effect o f in c re a s in g c o n c e ntra tio n s o f s a lm on c a lc ito n in ( sCT) o nC AMP a c c u m u la tio n in T 4 7 D , H x l a n d H x2 c e lls . T h e r e s u l t s a rep re s e n te d a s fm o l o f c AMP /2 4 -w e Il p la te . A , H x i (A) a n d H x2 (U ); B ,T 4 7D .

TABLE 2ED fo r c AMP response

1 m M EG TA

20 nM s C T

0 100 200S e c o n d s 300

400

F ig . 7 . E ffe c t o f 2 0 flM s a lm on c a lc ito nin ( sCT) o n in tra ce llu la r c a lc iumle ve ls in H xl c e lls . A , R e s u lts o b ta in e din b u ffe r c o n ta in in g 1 .8 m c a lc iu m ; B , re s u lts O b ta in e d in u ffe r c on ta in in g 0 .5 m c a lc iu m a n d 1m M E G T A .

0 0.032 0.1 0.32

s C T (n m )

E0 .U

CeED,

H umai Sm onrfl u

T4 7 D 2 .7 1 .2 0 .4 5 0 .2H xl 0 .0 8 0 . 0 1 0 . 0 6 0 .0 2H x2 0 .0 7 0 .0 1 0 .0 4 0 . 0 1

H xl

o _ _ _ _ _ _ o _ _ _ _ _ _ _ _ _ o . _ _ _ _ _ _ _ _ o T4 7 D

.1

to the calci tonin receptor gene to ampl i f y D NA samples i solatedf rom a series of human/rodent somatic cel l hybr ids that containonly one or tw o human chromosomes. T he resul ts, show n inFig. 10, show that a band of the expected si ze w as obtainedonly w i th the control human DNA and w i th DNA isolated f romthe somatic cell hybr id that contains human chr omosome 7.This indicates that the calci tonin receptor gene resides onchromosome 7.

Thi s l ocati on w as conf i rmed by f l uorescent in situ hybrid i-zati on (Fig. i i ). Our probes were a genom ic f ragment (70-100

1 10 100 1000s C T (n M)

F ig . 8 . Effect o f in c re a s in g c o n c e ntra tio n s o f s a lm on c a lc ito n in(sCT ) o nth e p r o d u c t n o f in o s ito l p h o s p h a te s in H xl a n d T4 7 D c e lls . T h e a ve ra g eb a c k g ro u n d le ve l fo r H x l w a s 2 7 8 c p m a n d fo r T 4 7 D w a s 1 3 5 c p m .

TAB LE 3ED f or i nosi tol PhOsphate response I n H xI cells

Agent EDflM

H um a n c a ic a to nin 9 .5 0 .7S a lm o n c a lc ito n in 6 1 .4


8/10

20

10

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

Calc i ton in Receptor Subtypes 253

W e found that the transfected insert-negative form of t

5)C,)C00.C, )5)

a-00U-

Bi n d i n g(P e rc e nt o f HxI)

F ig . 9 . Ma xim um ino sito l pho spha te ( IP) re s po n s e s to s alm o n c a lc ito n inde te rm ine d in a s e rie s o f tra n s fe c ta n ts tha t e xpre s s va ry ing le ve ls o f th ec a lc ito nin re ce pto r. The b ind ing c apa city o f e ac h tra ns fe cta ntw as de te r -m ine d in th e pre s e n c e o f s a tu ra ting am o un ts o f a l-hum a n c a lc ito n in .Th is v a lu e w a s no rm a liz e d to 1 0 c e lls a nd is e xpre s s e d a s th e pe rc e n t-a g e o f b ind ing d is p la ye d by Hxl c e lls .

Fig . 1 0 . P CR amp lific a tio n o f DNA Obta in e d fro m a pa ne l o f hum a n /ro de n t s o m a tic c e ll h ybrid s tha t e a c h c o n ta in o ne o r tw o hum a n c hro -m o s o m e s . Ea c h la ne c o rre spo nd s to a d iffe re nt hybrid ;anes 25 , 26 , a nd27, m o us e , C h in e s e ham s te r, a nd hum a n DNA, re s p e c tive ly .Lane 7 , ahybrid tha t c o n ta in s o n ly hum a n c hrom o s om e 7 .kb) containing a portion of the human calcitonin receptor genea n d a cen t r om er ic p r o b e sp ec if ic fo r ch r om o som e 7.

D i s c u s s i o nOu r r esu lt s d em on st r a t e t h a t t h e r e a r e tw o iso fo r m s o f t h e

human ca lc it on in r ecep t o r , w h ich d iffe r b y t h e p r esen ce orabsence of a 16-amino acid insert in the first intracellular loop.Subtypes of o t h er G protein-coupled receptors that are ex-pressed in a tissue-specific fashion and have different phar-macological properties have been reported (3). A fter confirmingt h a t b o t h fo r m s o f t h e r ecep t o r u n d er w en t eq u iva len t a m p lifi-

F ig . I 1 . Hum a n m e ta pha s e c hrom o s om e s pre a d hybrid iz e d w ith a g o -n om ic fra gm e n t o f th e h um a n c a lc ito n in re c e p to r g e ne a nd w ith ac e ntrom e nc pro b e s pe cific fo r c h rom o s om e 7 . a rge arrow s, cen tromer icla b e lin g o f c h rom o s om e 7 ;mall arrows, lo c atio n o f th e c alc ito nin re ce p-to r g e ne o n th e q a rm .cation using the PCR, we used this technique to examine trelative frequency of these two receptor subtypes in a varietyof tissues that express the calcitonin receptor. These includedkidney, whole fetal brain, hypothalamus, mammary gland, cer-ebral cortex, cerebellum, lung, stomach, placenta, ovary, anbone marrow. The insert-negati e form of the receptor appearsto be the most abundant form of the receptor and is expressedat a relatively constant level in all tissues that we examined.In contrast, the levels of expression of the insert-positive recep-tor ranged from being undetectable in stomach, fetal brain, acerebral cortex to constituting almost half of the transcriptsfound in ovary and placenta. This observation suggests ththe expression of this receptor isoform may be regulated intissue-specific manner or may be affected by various physiolog-i cal f actor s.

W e expressed the insert-negative form of the receptorBHK cells and characterized the transfectants w ith respectbinding and second messenger coupling. T he binding affinitiesfor human calcitonin and salmon calcitonin are the samethose of the endogenous receptor in T 47D cells and are vesimilar to the values reported for the insert-positive form ofthe receptor (10). T herefore, the binding properties ofreceptor are apparently notffected by the presence or absenceof the insert.


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254 Kuestner at aLreceptor couples to both the cA M P and phosphol ipase C path-w ays. A sim i lar observation haseen made f or the porcinecalci tonin receptor (27, 28). U nfortunatel y , a compari son w i ththe insert-posi ti ve f orm of the human receptor i s di f f i cul t tomake. Gorn e t a! . (10) reported that the insert-posi ti ve receptorcouples to the cA M P pathway at saturati ng amounts of salmoncalci tonin, but those authors did not look for phosphol ipase C-mediated events.

W e found that 10-100-f old lower levels of calci tonin arerequired to stimulate a cA M P response in the transfectants,compared w i th T47D cel l s. T his augmented cA M P responsemay be due to overexpression of the receptors in the transf ec-tants. H ow ever, the EDse values are identi cal f or H x l and H x2,despi te an 8-f old di f f erence in thei r receptor numbers. Further-more, Force e t a t. (27) f ound that the ED se for the recombinantporcine calci tonin receptor i s 40-f old low er than the EDse f orthe endogenous receptor expressed by L L C-PK 1 cel l s. In thatcase, both the transfectant and L L C-PK 1 cel l s express 300,000receptors/cel l , so the di f ference in the EDse values is not dueto a di f f erence in receptor numbers. I t i s possible that theaugmented cA M P response mediated by the transfected recep-tor occurs because the dow n-regulati on or desensi ti zati on ofthe recombinant receptor i s impai red in B HK cel l s. T his si tu-ati on occurs in yeast cel l s, w hich express a desensi ti zati on-resi stant f orm of the a factor receptor and are hyperresponsi veto pheromone action (29). Furthermore, a recombinant thyro-tropin receptor expressed in Chinese hamster ovary cel l s doesnot undergo homologous desensi ti zati on as i t does in thyroidcel l s, suggesting that a ti ssue-speci f i c factor i s requi red f or thi sprocess (30). A study of the dow n-regulati on and desensi ti za-ti on of the human recombinant calci tonin receptor in B HKcel ls is underw ay .

W e also f ound that stimulati on of the phosphol ipase C path-w ay in the transf ectants appears to be di rectl y correlated w i ththe receptor number and requi res approx imatel y 100 timesmore calci tonin than the cA M P response. T he level of cal ci -tonin requi red f or a phosphol ipase C response (ED se 5 nM )i s si gni f i cantl y hi gher than normal plasma level s (2-10M ) andeven exceeds the peak plasma levels obtainedafter calcitonini njecti on therapy (100 pM ) (1). These observations may indicatethat the phosphol ipase C pathway does not play a physiologicalrole in calci tonin signal i ng. H ow ever, the second messengercoupl i ng of the calci tonin receptor can be modulated by avari ety of f actors. For example, i n L L C-PK 1 k idney cel ls cal -ci toni n preferential l y stimulates the cA M P pathw ay duri ng theG2 phase of the cel l cycle, w hereas protein k inase C is acti v atedduring S phase (2). Furthermore, the abi l i ty of cal ci tonin tostimulate i nternal cal cium level s i n osteoclasts can be signi f i -cantl y augmented by elevating ex tracel l ular calcium levels f rom2mMto6mM(26 ).

In conclusion, our w ork show s that there are subtypes of thehuman cal ci toni n receptor. W e are currentl y investi gatingw hether these subtypes are generated by al ternati v e spl i cing orare products of tw o independent genes. The physiological sig-ni f i cance of these subtypes remains to be establ i shed.Ac k n ow l e d gm e n t s

The authors thank D r . D i ane D urnain for her suggestions concerning themanuscript.Re f e r en c e s1. A zria, M . The Co icitoniris: Physio logy and Pharmacology. K arger, B asel

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11. Sexton, P. M ., S. H oussam i , J. M . H i l ton, L M . O K eef fe, R J. Center,T . G i l l espie, P. D arcy , and D . M . Findlay . I denti f i cation ofbrai n i soformsthe rat calci toni n receptor. M oL E nd oc ri no L 7:815-821 (1993).

12. A lbrandt, K ., E. M ul l , E . M . G . B rady , J. H erich, C . X . M oore,nd K .Beaumont. M olecular cloning of tw o receptors f rom rat brain w i th hi ghaf f ini ty f or salmon calci tonin. FEBS Lea . 325:225-232 (1993).

13. Y ami n, M ., M . R Flannery , D . Rapp, A . H . Gore, S. M . K rane, and S. RGoi dr i ng. A na1ysi of a unique murine brain calci tonin receptor (C T R ) cD NAand prel im inary character i zation of the marineCT R gene: ev idence for theex istence of f unctional l y dist inct i soforms of the CTR.. Bone Minera l Res.8:S129 (1993).

14 . K u r o k a w a , M ., V . P . M ich ela n g eli, a n d D . M . F in d la y . I n d u ct ionf c al ci to ni nreceptor expression by glucocort i coids in T 47D human breast cancer cel l s.EndocrinoL 130:321-326 (1991).

15. Chi rgw in, J. M . I sol ation of biol ogi cal l y acti v e r ibonucleic acid f rom souenriched i n r ib o nu c le as e. Biochemistry 18: 52-94 (19 79 ).

16. A v iv , H ., and P. L eder . Pur i f i cati on of biological l y acti v e globin messenR NA b y ch r om atog r a p h y on o ligo t h ym id y lic a c id -cellu lo se . P roc . Na tL Acad.Sc i. U SA 69:1408-1421 (1972).

17. O H are, P. J., and D . V enezia. PR IM EGEN , aool f or designing primersf rom mul tip le al ignments. CABIOS 7:533-534 (1991).

18. Sanger, F., S. N i ck l en, and A . R . Coul son. DN A sequenci ng w i thterminat ing inhibitors. Proc. NatL Acad. Sci USA74:5463-5467 (1977).

19. Graham, F. L ., and A . J. van der Eb. A newechnique f or the assay ofinfec t iv ity of hum an adenov irus 5 DN A . Virology 52:456-467 (1973).

20. M cPherson, G. A . A nalysis of radiol i gand bi ndi ngxp er im en t s: a co llect ionof computer programs for theBM PC. J. PharmacoL Methods 14:213-228(1985).

21. Stroop, S. D ., D . L Thompson, R . E. K uestner , and E. E. M oore. A rbinant human calci tonin receptor functions as an ex tracel lu lar calsensor. J. BiOL Chem. 268:19927-19930 (1993).

22. D urnam , D . M ., J. C. M enninger, S. H . Chandler, P. P. Sm i th, andM cDougal l . A f ragi le si te inhe human U2 sm al l nucl ear RN A g en e cl usteris revealed by adenov i rus type 12 inf ecti on. MoL CelL BIOL 8:1863-1867(1988).

23. Palm i ter , R D ., S. D . F indley , T . E. W hi tmore, and D . M . D urnam . M Ta brain-speci f i c member of the metal l othi onein gene fam i l y .ro c. NO2L Acad.Sc i. U SA 89:6333-6337 (1992) .

24. Raue, F., H . G. Schneider, A . Z ink , and R. Z iegler. A ction of calci toninrel ated peptide at the cal ci tonin receptor of the T 47D cel l l i ne.o rm. Metob.Res. 19:563-564 (1987).

25. M oonga, B . S., A . S. M . Towhidul A lam , P. J. R. B ev i s, F. A valdi , i t SonC . L .-H . H u a n g , a n d M . Z a id i. R egu la t ion ofcy t oso lic free calcium i n i so lat edr a t osteoclasts b y c al ci t om n . J . E nd oc ri no L 132:241-249 (1992).

26. M algarol i , A ., J. M eldolesi , A . Z . Zal lone, and A . Teti . Control of cy tofree calcium in rat and chicken osteoclasts: the role of ex tracel lu lar calcia n d c alc it on in . J. BiOL Chem. 264 :14 342-1 4347 (19 89) .

27. Force, T ., J. V . Bonventre, M . R Flannery , A . H . Gore, M . Y am in, andGoldring. A cloned porcine renal calci toni n receptor couples to adeny ly lcyclase a n d p h osp h olip a se C .Am. J. P hys io L 262 :F1 110 -F1115 (1992).

28. Chabre, 0., B . R Conk l in, H . Y . L in, H . F. L odish, E. W i lson, H . E. I vCatanzari ti , B . A . H emmings, and H . R B ourne. A recombinant calci ton


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Calci ton in R ecep to r S ub types 2 55receptor independ en tly stim ula tes 3 ,5 -cyc ic adeno sine monopho sph ate and th yro id al euk aryotic c ells p rov ide s evid en ce th at homo logousdesens i t iza t ionC a2 ino aito l phospha te sig na ling p athw ays. Mo!. E n d O C r I n O L 6 :551-556 to thy rotropin stim ulatio n requires a ce ll-spec ific facto r.ndocrinology(1992) . . 127 :1240-1244 (1990).

2 9 . R enek e, J. E ., K . J. B lum e r, W . E . C ou rch esn e, an d J. T horner. T he carb oxy -___ _____ ____ _____ ____ ____ _____ ____ _____ ____ ___term ina l segm en t o f th e yea sta facto r receptor is a regulato ry dom a in . Cell55:221-234 (1988 ). S end reprin t requ ests to : Emma E . M oo re, Z ym oG enetic s, 1201 Eas tlakeve .

30. Chazenb alk , G . D ., Y . N ag ayam a , K . D . K a ufm an , and B . R apopo rt T he E ., S eattle , W A 98102.fun ctiona l expre ssion o f r ec om b in an t hum an thy rotrop in re cep tors in non-

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