g and phospholipase c-b: turn on, q turn off, and do it fast 2011 science... · phospholipase c-β3...

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PERSPECTIVE www.SCIENCESIGNALING.org    00 Month 2010    Vol X Issue X  DocX        1 How does a signaling enzyme turn off one of its own activators and still respond? And why? The structure of a complex of phospholipase C-β3 (PLC-β3) and its up- stream activator Gα q helps answer these questions (1). It also corrects a misconcep- tion, describes some interesting convergent evolution, suggests a general structure for recognizing G proteins, poses some hard mechanistic questions, and provides a tool to help answer them. First, some background. In animals, phospholipase Cs (PLCs) degrade phospha- tidylinositol 4,5-bisphosphate, an important signaling molecule, and create two second messengers: diacylglycerol and inositol 1,4,5-trisphosphate (which triggers the re- lease of another second messenger, Ca 2+ , and is also the metabolic precursor of the inositol polyphosphate second messengers). The six mammalian PLC subfamilies re- spond to diverse inputs. The PLC-βs are stimulated by the Gα subunits of the G q heterotrimeric G proteins, Gβγ subunits re- leased by the G i ’s, Ca 2+ and, for one isoform, the small guanosine triphosphate (GTP)– binding protein Rac (2). In addition, the PLC-βs are GTPase-acti- vating proteins (GAPs) for the Gα q proteins that activate them (Fig. 1). G protein sig- naling is based on a cycle of GTP binding and hydrolysis. GTP activates the G protein and allows it to activate its downstream ef- fectors; hydrolysis of GTP to guanosine di- phosphate (GDP) causes deactivation and terminates signaling. Receptors act by cata- lyzing release of GDP and binding of GTP to create active, GTP- bound G protein. GAPs accelerate the deactivation limb of the cycle, which would otherwise be very slow (10 s to 3 min). GAPs can in- hibit signal output by shifting the balance of G protein to the GDP-bound state. GAPs also are important for pro- moting fast signal termination when re- ceptor is deactivated, and they do so with- out inhibiting signal- ing while receptor is active. How this works is not clear (1, 3). Why should an effector turn off the G protein that activates it? The structure of a complex of Gα q and PLC-β3, described by Waldo et al. (1), ex- plains the mechanism of PLC-β’s GAP ac- tivity and suggests how it can function with- out turning off the signal that it listens to. Waldo et al. solved the structure of a complex of Gα q and the globular core of PLC-β3. The PLC-βs are composed of two pieces, the core and a long, C-terminal coiled coil (CC) domain (2) (Fig. 2). This core, which is common to all mammalian PLCs, is composed of a pleckstrin homol- ogy (PH) domain, four EF hands, a trios- ephosphate isomerase (TIM) barrel that includes the catalytic site, and a C2 do- main (2)(Fig. 2). The first surprise was that the PLC-β core still responds to Gα q after removal of the CC domain, which is the salient marker of the β class of PLCs and which was thought to bind Gα q (4, 5). Re- moval or mutation of the CC domain blocks stimulation of PLC-β by Gα q in cells and several in vitro assays systems (5-7), and the isolated CC domain both displays some G q GAP activity (8) and inhibits PLC-β activa- tion by G q (8, 9). Waldo et al. showed that the PLC-β3 core remains sensitive to stimu- lation by Gα q and retains G q GAP activity. The affinity of the truncated PLC-β3 for G q is similar to that of the full-length protein, according to surface plasmon resonance, al- though the affinity measured in this assay is lower by a factor of about 100 than that estimated in membrane-based experiments (10). Waldo et al. reasonably infer that the coiled coil helps to anchor PLC-β to the membrane surface, thus increasing its local concentration, and that this effect is neces- sary in cells (11). It does not explain why removal of the CC domain inhibits stimula- tion only by Gα q but not by Gβγ. Clearly, other activities of the CC domain must be reexamined. STRUCTURAL BIOLOGY Ga q and Phospholipase C-b: Turn On, Turn Off, and Do It Fast Elliott M. Ross,* *Corresponding author. Phone, 214-645-6134. E-mail, [email protected] Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 For- est Park Road, Dallas, TX 75390–9041, U.S.A. CREDIT: Y. HAMMOND/SCIENCE SIGNALING CREDIT: Y. HAMMOND/SCIENCE SIGNALING Heterotrimeric G proteins and G protein–coupled receptors represent conserved protein families with origins in the prokaryotes, but the various G protein– regulated effectors are heterogeneous in structure and function. The effectors apparently evolved ways to listen to G proteins late in their evolutionary histo- ries. The structure of a complex between the effector protein phospholipase C-b3 (PLC-b3) and its activator, Ga q , suggests that several effectors independently evolved a structurally similar helix-turn-helix segment for G protein recognition. PLC-bs are also guanosine triphosphatase (GTPase) activating proteins (GAPs) for the G q that activates them. In a second example of convergent evolution, the GAP activity of these proteins depends on a flexible asparagine-containing loop that resembles the GAP site on RGS proteins, another family of G protein GAPs. Together, these two sites are proposed to cooperate to enable fast binding to acti- vated Ga q , followed by fast deactivation. This cycle allows rapid sampling of the ac- tivation state of G q -coupled receptors while providing efficient signal transduction. Fig. 1. The signal output of a G protein module is proportional to the  concentration of the active Gα-GTP species and represents the bal- ance of the rates of receptor-catalyzed GDP/GTP exchange (activa- tion) and GAP-accelerated GTP hydrolysis (deactivation). In the case  of the PLC-βs, the effector is itself the GAP. Waldo et al. (1) describe  how PLC-β can repeatedly engage and disengage specific sites on  Gα q during rapid cycling of the GTPase reaction. G q -GDP G q *-GTP GDP GTP PLC-β Pi (GAP) Receptor

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Page 1: G and Phospholipase C-b: Turn On, q Turn Off, and Do It Fast 2011 Science... · phospholipase C-β3 ... and Phospholipase C-b: Turn On, Turn Off, and Do It Fast ... nus and truncated

P E R S P E C T I V E

www.SCIENCESIGNALING.org    00 Month 2010    Vol X Issue X  DocX        1

How does a signaling enzyme turn off one of its own activators and still respond? And why? The structure of a complex of phospholipase C-β3 (PLC-β3) and its up-stream activator Gα

q helps answer these

questions (1). It also corrects a misconcep-tion, describes some interesting convergent evolution, suggests a general structure for recognizing G proteins, poses some hard mechanistic questions, and provides a tool to help answer them.

First, some background. In animals, phospholipase Cs (PLCs) degrade phospha-tidylinositol 4,5-bisphosphate, an important signaling molecule, and create two second messengers: diacylglycerol and inositol 1,4,5-trisphosphate (which triggers the re-lease of another second messenger, Ca2+, and is also the metabolic precursor of the inositol polyphosphate second messengers). The six mammalian PLC subfamilies re-spond to diverse inputs. The PLC-βs are stimulated by the Gα subunits of the G

q

heterotrimeric G proteins, Gβγ subunits re-leased by the G

i’s, Ca2+ and, for one isoform,

the small guanosine triphosphate (GTP)–binding protein Rac (2).

In addition, the PLC-βs are GTPase-acti-vating proteins (GAPs) for the Gα

q proteins

that activate them (Fig. 1). G protein sig-naling is based on a cycle of GTP binding

and hydrolysis. GTP activates the G protein and allows it to activate its downstream ef-fectors; hydrolysis of GTP to guanosine di-phosphate (GDP) causes deactivation and terminates signaling. Receptors act by cata-lyzing release of GDP and binding of GTP to create active, GTP-bound G protein. GAPs accelerate the deactivation limb of the cycle, which would otherwise be very slow (10 s to 3 min). GAPs can in-hibit signal output by shifting the balance of G protein to the GDP-bound state.

GAPs also are important for pro-moting fast signal termination when re-ceptor is deactivated, and they do so with-out inhibiting signal-ing while receptor is active. How this works is not clear (1, 3). Why should an effector turn off the G protein that activates it? The structure of a complex of Gα

q and

PLC-β3, described by Waldo et al. (1), ex-plains the mechanism of PLC-β’s GAP ac-tivity and suggests how it can function with-out turning off the signal that it listens to.

Waldo et al. solved the structure of a complex of Gα

q and the globular core of

PLC-β3. The PLC-βs are composed of two pieces, the core and a long, C-terminal coiled coil (CC) domain (2) (Fig. 2). This core, which is common to all mammalian PLCs, is composed of a pleckstrin homol-ogy (PH) domain, four EF hands, a trios-ephosphate isomerase (TIM) barrel that includes the catalytic site, and a C2 do-main (2)(Fig. 2). The first surprise was that the PLC-β core still responds to Gα

q after

removal of the CC domain, which is the salient marker of the β class of PLCs and which was thought to bind Gα

q (4, 5). Re-

moval or mutation of the CC domain blocks stimulation of PLC-β by Gα

q in cells and

several in vitro assays systems (5-7), and the isolated CC domain both displays some G

q

GAP activity (8) and inhibits PLC-β activa-tion by G

q (8, 9). Waldo et al. showed that

the PLC-β3 core remains sensitive to stimu-lation by Gα

q and retains G

q GAP activity.

The affinity of the truncated PLC-β3 for Gq

is similar to that of the full-length protein, according to surface plasmon resonance, al-though the affinity measured in this assay is lower by a factor of about 100 than that estimated in membrane-based experiments (10). Waldo et al. reasonably infer that the

coiled coil helps to anchor PLC-β to the membrane surface, thus increasing its local concentration, and that this effect is neces-sary in cells (11). It does not explain why removal of the CC domain inhibits stimula-tion only by Gα

q but not by Gβγ. Clearly,

other activities of the CC domain must be reexamined.

S T R U C T U R A L B I O L O G Y

Gaq and Phospholipase C-b: Turn On, Turn Off, and Do It FastElliott M. Ross,*

*Corresponding author. Phone, 214-645-6134. E-mail, [email protected]

Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 For-est Park Road, Dallas, TX 75390–9041, U.S.A.

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Heterotrimeric G proteins and G protein–coupled receptors represent conserved protein families with origins in the prokaryotes, but the various G protein–regulated effectors are heterogeneous in structure and function. The effectors apparently evolved ways to listen to G proteins late in their evolutionary histo-ries. The structure of a complex between the effector protein phospholipase C-b3 (PLC-b3) and its activator, Gaq, suggests that several effectors independently evolved a structurally similar helix-turn-helix segment for G protein recognition. PLC-bs are also guanosine triphosphatase (GTPase) activating proteins (GAPs) for the Gq that activates them. In a second example of convergent evolution, the GAP activity of these proteins depends on a flexible asparagine-containing loop that resembles the GAP site on RGS proteins, another family of G protein GAPs. Together, these two sites are proposed to cooperate to enable fast binding to acti-vated Gaq, followed by fast deactivation. This cycle allows rapid sampling of the ac-tivation state of Gq-coupled receptors while providing efficient signal transduction.

Fig. 1. The signal output of a G protein module is proportional to the concentration of the active Gα-GTP species and represents the bal-ance of the rates of receptor-catalyzed GDP/GTP exchange (activa-tion) and GAP-accelerated GTP hydrolysis (deactivation). In the case of the PLC-βs, the effector is itself the GAP. Waldo et al. (1) describe how PLC-β can repeatedly engage and disengage specific sites on Gαq during rapid cycling of the GTPase reaction.

Gq-GDP G

q*-GTP

GDP GTP

PLC-β

Pi(GAP)

Receptor

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P E R S P E C T I V E

www.SCIENCESIGNALING.org    00 Month 2010    Vol X Issue X  DocX        2

The structure also describes the Gα

q-PLC-β3 interface: three

separate segments of PLC-β3 that bind Gα

q over a large con-

tact area (Fig. 2). Both a loop at the N terminus of the C2 domain and a loop off an EF hand bind Gα

q near its switch regions. The

switches are short segments of Gα subunits near the GTP bind-ing site that change conformation upon GTP binding and hydro-lysis. In addition, a helix-turn-helix extension at the C terminus of the C2 domain wraps around the edge of the Gα

q to provide

an additional large contact area. The contact sites at both ends of the C2 domain are required for activation of PLC-β3 by Gα

q;

the loop off the EF hand medi-ates G

q GAP activity. Creation of

this interface depends on the pre-vious dissociation of Gβγ from Gα

q because the PLC-β3 and

Gβγ contact sites on Gαq over-

lap substantially (1). Although Gα

q and Gβγ (derived from a G

i

heterotrimer) bind PLC-β3 si-multaneously and synergistically (12), they may be only loosely at-tached to each other in the three-protein complex, if they touch at all. This situation is reminiscent of the binding of Gα

q and Gβγ to

a protein kinase, where the two subunits are substantially distant from each other (13).

Strikingly, none of the Gαq-

PLC-β3 interface is anywhere near the PLC active site. This leads to the first unanswered question: How exactly are the PLCs activated? Results from several groups indicate that an autoinhibitory loop in the middle of the TIM barrel blocks the active site, and its removal by proteolysis or mutation is sufficient to activate PLC-β, -γ, -δ, and -ε (14-17). In PLC-γ, which is stimu-lated by phosphorylation of a tyrosine resi-due in this loop, intraloop binding of an SH2 domain to the phosphotyrosine reconfigures the loop to relieve autoinhibition (18). For the PLC-βs and others subfamilies, howev-er, this loop is small and there is no indica-tion that any of the allosteric activators drive a propagated change in structure to move the loop out of the way. Superposition of crystal structures shows that activated and

basal proteins are strikingly similar, also arguing against a global restructuring (at least in crystals) (1, 14, 19). To explain this conundrum, Sondek’s group suggested that Rac, Gα

q, or Gβγ might all activate PLC-β

proteins simply by binding them tightly to the plasma membrane surface with no intrinsic conformational change. There, negatively charged lipids might shove the negatively charged loop out of the way (14). Ca2+, which also activates PLC, might work similarly. Unfortunately, there is no direct support for this mechanism, and many of the PLCs are stably bound to membranes without activation. This suggestion will be hard to test. An intuitively supporting result comes from a test of the importance of the

helix-turn-helix extension of the PLC-β3 C2 domain. When this segment from PLC-β3 was appended to the C terminus of PLC-δ1, which is not regulated by Gα

q, the resulting chimera

was stimulated about two-fold in response to G

q and a G

q-coupled

receptor. Such activation is mod-est compared to the eight-fold stimulation of wild-type PLC-β3 in the same assay, and only a tiny fraction of the 400-fold response to saturating activated Gα

q (12).

Nevertheless, this result sug-gests that all of the mammalian PIP

2-specific PLCs can be acti-

vated in fundamentally the same way.

The helix-turn-helix segment also seems to be a wonderful ex-ample of convergent evolution. Gα and Gβγ subunits and the G protein–coupled receptors are all members of evolutionarily conserved families with origins in the prokaryotes. In contrast, the effector proteins that execute their intracellular signals, such as the PLC-βs, are a wildly het-erogeneous group: assorted en-zymes, channels and transport-ers; cytosolic, nuclear, both and integral and peripheral mem-brane proteins. Mechanisms of effector activation are similarly heterogeneous, but these diverse regulatory proteins each man-aged to evolve some way to re-spond to G proteins. Waldo et al. show that the helix-turn-helix interface of PLC-β3 with Gα

q

is remarkably similar in over-all structure to those of several other Gα-effector complexes: adenylyl cyclase with Gα

s, G protein–coupled receptor kinase 2

(GRK2) and p63Rho guanine nucleotide exchange factor (GEF) with Gα

q, and the

γ subunit of cyclic guanosine monophos-phate (GMP) phosphodiesterase with Gα

t.

The amino acid sequences around the helix-turn-helix interface are not at all conserved (except for lack of charge), but each evolved its regulatory interactions with Gα subunits through modest changes in a similarly shaped surface.

The final contribution of this paper is to demonstrate the structural basis of the G

q

GAP activity of PLC-β. The authors noted

Fig. 2. (A)  Domain  map  of  PLC-β3,  color  coded  by  domain.  (B) Structure  of  the  PLC-β3-Gαq  complex  (PDB  3OHM). The  figure  is oriented such that the plasma membrane is at the top. The C termi-nus and truncated N terminus of Gαq (darker green), the two catalytic histidine residues of the PLC (purple), and the two ends of the un-structured autoinhibitory loop (rose) are all expected to be at or near the membrane surface. The asparagine required for Gq GAP activity is shown in red spheres. The flexible loops that contain the GAP site (orange) and that link the helix-turn-helix (pink-purple) to the C2 do-main (blue) are shown as thick spaghetti. Both loops are disordered in crystals of PLC-β2 alone (19). The Gαq contact site between the TIM barrel and C2 domains is bright magenta.

PH

EF TIM C2

CC

A

B

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P E R S P E C T I V E

www.SCIENCESIGNALING.org    00 Month 2010    Vol X Issue X  DocX        3

that an asparagine residue on a loop off one of the EF hand domains contacts switch I on Gα

q in a way reminiscent of the contact of

Gα subunits with RGS proteins, the other class of G protein GAPs. They show that mutating this asparagine on PLC-β3 blocks G

q GAP activity. More convergent evolu-

tion! Although there is no sequence similar-ity between the PLC-βs and RGS proteins, they both carry flexible loops that place as-paragines at just the right spot to stabilize residues in Gα that are important for GTP hydrolysis. This interaction leads to accel-eration of GTP hydrolysis by a factor of more than 1000 (20). It thus seems easy to evolve G protein GAP activity: just give an asparagine some flexibility at the right place on the surface of the effector. The down side is that, with only a one-residue “consensus sequence,” it will be hard to identify new GAPs by sequence alone. There are prob-ably a lot more out there.

What is the physiologic effect of giving an effector the ability to turn off its G protein activator? In the Drosophila eye, where rho-dopsin activates a G

q-PLC-β pathway rather

than the Gt-phosphodiesterase pathway of

mammalian photoreceptors, the asparagine mutation markedly delayed recovery after exposure to a light flash (1). The delay pre-sumably reflects the longer time needed for G

q to hydrolyze bound GTP and return to

the inactive state. This result is similar to that of mutating the asparagine of the mam-malian photosensory GAP, a distinct RGS protein that deactivates G

t (21). This new

result strongly argues that the GAP activity of PLC-β is crucial for physiologically fast signaling. The asparagine mutation did not change signal amplitude, however, indicat-ing that the GAP activity of PLC does not substantially inhibit signal throughput. The GAP-less PLC-β also provides a valuable tool for studying the dynamics and regula-tion of the receptor-G

q-PLC-β pathways in

other cells.So how does all this work in real time?

Why design a signaling system where a downstream component deactivates its own activator? If it is only to achieve a fast turn-off rate, why not evolve a faster GTPase in Gα

q? The answer may involve what engi-

neers call sampling interval. The Gq-PLC-β

system allows a long initial activation life-time for G

q-GTP so that it can find PLC-β.

Then fast GTPase cycling takes over, and Gq-

PLC-β samples receptor activation with ~50

msec resolution (20). When receptor turns off, so does the PLC. Waldo et al. end with a model for how the contacts between PLC-β and Gα

q are made and broken during the GT-

Pase cycle. The crucial helix-turn-helix seg-ment is flexible in isolated PLC-β (14), and that flexibility may increase the chance of encountering a long-lived activated Gα

q. The

asparagine that loops off the EF hand domain then swings in to promote GTP hydrolysis, and the complex relaxes or may entirely dissociate. The authors’ fishing metaphor is “catch and release.” The catch by the helix-turn-helix may be “foul-hooking,” away from functional sites, but the release still works.

How does the receptor keep up with the fast hydrolysis rate? Waldo et al. cite “kinet-ic scaffolding”: the idea that, with fast hy-drolysis, the receptor simply does not have time to dissociate from Gα

q-GTP, and hence

is able to quickly reactivate Gq once the

GAP turns it off (3,10). It is also likely that the GAP somehow potentiates receptor-G

q

coupling (3, 22). We clearly need real-time dynamic information about these processes. The new structure facilitates studying these dynamics in an intelligent way and provides useful tools for the effort.

References and Notes  1.  G. L. Waldo, T. K. Ricks, S. N. Hicks, M. L. Cheev-

er, T. Kawano, K. Tsuboi, X. Wang, C. Montell, T. Kozasa, J. Sondek, T. K. Harden, Kinetic scaffold-ing mediated by a phospholipase C-β and Gq sig-naling complex. Science 330, 974–980 (2010). 

  2.  M.  J.  Rebecchi,  S.  N.  Pentyala,  Structure,  func-tion,  and  control  of  phosphoinositide-specific phospholipase  C.  Physiol. Rev.  80,  1291–1335 (2000).

  3.  E.  M.  Ross,  Coordinating  speed  and  amplitude in G-protein signaling. Curr. Biol. 18, R777–R783 (2008). 

  4.  A. U. Singer, G. L. Waldo, T. K. Harden, J. Sondek, An unique fold of phospholipase C-β mediates di-merization and interaction with Gaq. Nat. Struct. Biol. 9, 32–36 (2002). 

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 23.  I thank the members of my group for comments on this essay. Work from my laboratory was sup-ported  by  NIH  grant  GM0303055  and  Welch Foundation grant I-0982.

10.1126/scisignal.2001798

Citation: E. M. Ross, Gαq and phospholipase C-β: Turn on, turn off, and do it fast. Sci. Signal. 4, pe5 (2010).