Activation of phospholipase C-epsilon by heterotrimeric G protein betagamma-subunits

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.     

Crystal structure of Rac1 bound to its effector phospholipase C-beta2

Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-beta2 (PLC-beta2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-beta2 to optimize its orientation for substrate membranes. Gbetagamma also engages the PH domain to activate PLC-beta2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-delta, the PH domain of PLC-beta2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1-PLC-beta2 complex reveals determinants that dictate selectivity of PLC-beta isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-beta2 abrogate its stimulation by Rac1 but not by Gbetagamma, allowing for functional dissection of this integral signaling node.     

The pleckstrin homology domain of phospholipase C-beta(2) links the binding of gbetagamma to activation of the catalytic core

Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.     

Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits

Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.     

Regulator of G-protein signaling 3 (RGS3) inhibits Gbeta1gamma 2-induced inositol phosphate production, mitogen-activated protein kinase activation, and Akt activation

Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which Galpha(i) and Galpha(q) hydrolyze GTP to GDP, thereby limiting the duration in which GTP-Galpha(i) and GTP-Galpha(q) can activate effectors. Since GDP-Galpha subunits rapidly combine with free Gbetagamma subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of Gbetagamma subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind Gbeta(5) in the absence of a Ggamma subunit, RGS proteins are not known to directly influence Gbetagamma signaling. Here we show that RGS3 binds Gbeta(1)gamma(2) subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with Gbeta(1)gamma(2) inhibits Gbeta(1)gamma(2)-induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of Gbeta(1)gamma(2) signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect Gbeta(1)gamma(2) signaling in these assays. Consistent with the in vivo results, RGS3 inhibits Gbetagamma-mediated activation of phospholipase Cbeta in vitro. Thus, RGS3 may limit Gbetagamma signaling not only by virtue of its GTPase-activating protein activity for Galpha subunits, but also by directly interfering with the activation of effectors.     

Gbeta 5gamma 2 is a highly selective activator of phospholipid-dependent enzymes

In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.     

Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2

A critical role of the Gbetagamma dimer in heterotrimeric G-protein signaling is to facilitate the engagement and activation of the Galpha subunit by cell-surface G-protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and the Gbetagamma dimer has not previously been available. Here, we describe the structural determinants of Gbeta1gamma2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by X-ray crystallography. The structure reveals that several critical residues within PTH1R contact only Gbeta residues located within the outer edge of WD1- and WD7-repeat segments of the Gbeta toroid structure. These regions encompass a predicted membrane-facing region of Gbeta thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gbeta1 leads to a selective loss of function in receptor/heterotrimer coupling while preserving Gbeta1gamma2 activation of the effector phospholipase-C beta.     

Gbetagamma subunit combinations differentially modulate receptor and effector coupling in vivo

In vitro, little specificity is seen for modulation of effectors by different combinations of Gbetagamma subunits from heterotrimeric G proteins. Here, we demonstrate that the coupling of specific combinations of Gbetagamma subunits to different receptors leads to a differential ability to modulate effectors in vivo. We have shown that the beta(1)AR and beta(2)AR can activate homomultimers of the human inwardly rectifying potassium channel Kir 3.2 when coexpressed in Xenopus oocytes, and that this requires a functional mammalian Gs heterotrimer. Modulation was independent of cAMP production, suggesting a membrane-delimited mechanism. To analyze further the importance of different Gbetagamma combinations, we have tested the facilitation of Kir 3.2 activation by betaAR mediated by different Gbetagamma subunits. The subunits tested were Gbeta(1,5) and Ggamma(1,2,7,11). These experiments demonstrated significant variation between the ability of the Gbetagamma combinations to activate the channels after receptor stimulation. This was in marked contrast to the situation in vitro where little specificity for binding of a Kir 3.1 C-terminal GST fusion protein by different Gbetagamma combinations was detected. More importantly, neither receptor, although homologous both structurally and functionally, shared the same preference for Gbetagamma subunits. In the presence of beta(1)AR, Gbeta(5)gamma(1) and Gbeta(5)gamma(11) activated Kir 3.2 to the greatest extent, while for the beta(2)AR, Gbeta(1)gamma(7), Gbeta(1)gamma(11,) and Gbeta(5)gamma(2) produced the greatest responses. Interestingly, no preference was seen in the ability of different Gbetagamma subunits to facilitate receptor-stimulated GTPase activity of the Gsalpha. These results suggest that it is not the receptor/G protein alpha subunit interaction or the Gbetagamma/effector interaction that is altered by Gbetagamma, but rather that the ability of the receptor to interact productively with the Gbetagamma subunit directly and/or the G protein/effector complex is dependent on the specific G protein heterotrimer associated with the receptor.     

Phospholipase C-beta1 directly accelerates GTP hydrolysis by Galphaq and acceleration is inhibited by Gbeta gamma subunits.

Phospholipase C-beta, the principal effector protein regulated by Galphaq, has been shown to increase the agonist-stimulated, steady-state GTPase activity of Gq in proteoliposomes that contain both heterotrimeric Gq and m1 muscarinic receptor. We now use a moderately stable complex of R183C Galphaq bound to GTP to show that PLC-beta1 acts directly as a GTPase-activating protein (GAP) for isolated Galphaq in a membrane-free system. PLC-beta1 accelerated the hydrolysis of GalphaqR183C.GTP up to 20-fold. The Km was 1.5 nM, which is similar both to the EC50 with which R183C and wild type Galphaq activate PLC-beta1 and to the EC50 with which PLC-beta1 acts as a Gq GAP in the vesicle-based assay. The Galphaq GAP activity of RGS4 can also be quantitated by this assay; it accelerated hydrolysis of bound GTP about 100-fold. The Gq GAP activities of both PLC-beta1 and RGS4 are blocked by Gbeta gamma subunits, probably by a competitive mechanism. These data suggest either that the Gbeta gamma subunits are not continuously required for receptor-catalyzed GDP/GTP exchange during steady-state GTP hydrolysis or that GAPs, either PLC-beta or RGS proteins, can substitute for Gbeta gamma in this set of reactions.     

PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.     

Coordinate expression of the alpha and beta subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

Individual cell types express a characteristic balance between heterotrimeric G protein alpha and betagamma subunits, but little is known about the regulatory mechanism. We systemically examined the regulatory mechanism in CHO cells. We found that expression of Galphas, Galphai2, and Galphaq proteins increased in direct proportion to the increase of Gbeta1gamma2 overexpressed transiently. Expression of Gbeta protein also increased following overexpression of Galphas, Galphai2, and Galphaq. The Gbetagamma overexpression stimulated degradation of Gbeta in contrast to reduction of Galphas degradation. We conclude that coordinate expression of the G protein subunits involves regulation of protein degradation via proteasome in CHO cells.     

Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.     

Enhancement of pheromone response by RGS9 and Gbeta5 in yeast

The G-protein gamma-subunit-like (GGL) domain present within a subfamily of RGS proteins binds specifically to Gbeta5. This interaction and resulting biological effect impacts the standard model of heterotrimeric G-protein signaling. It has been hypothesized that the RGS/Gbeta5 may potentially substitute for Gbetagamma in the heterotrimeric complex. Saccharomyces cerevisiae pheromone responsive mating signaling pathway is primarily driven by Gbetagamma. We evaluated GGL containing RGS9 and RGS7 for functional complementation in a RGS (sst2Delta) knockout yeast strain. The potential of Gbeta5 to augment the function of these RGS proteins was also evaluated. While Gbeta5 had no effect on RGS7, coexpression of Gbeta5 with RGS9 enhanced cell cycle arrest, suggesting that under certain conditions, RGS9 and Gbeta5 may possibly function as betagamma dimer. Furthermore, we demonstrate that Gbeta5 can complement a ste4Delta, the yeast beta-subunit, thus providing the first evidence of functional complementation of a mammalian Gbeta.     

A docking site for G protein βγ subunits on the parathyroid hormone 1 receptor supports signaling through multiple pathways

The G protein-coupled receptor for PTH and PTH-related protein (PTH1R) signals via many intracellular pathways. The purpose of this work is to investigate a G protein binding site on an intracellular domain of the PTH1R. The carboxy-terminal, cytoplasmic tail of the PTH1R fused to glutathione-S-transferase interacts with Gi/o proteins in vitro. All three subunits of the heterotrimer interact with the receptor C-tail. Activation of the heterotrimeric complex with GTPgammaS has no effect on Gbetagamma interactions, but markedly disrupts binding of the Galphai/o subunits to the receptor tail, suggesting that direct Gbetagamma binding indirectly links Galpha subunits to this region of the receptor. Gbetagamma subunits alone bind the C-tail with an affinity that is comparable to the heterotrimeric G protein complex. G protein complexes consisting of Galphashis6-beta1gamma2 and Galphaqhis6-beta1gamma2 also interact with the PTH1R tail in vitro. The Gbetagamma interaction domain is located on the juxta-membrane region of the tail between amino acids 468 and 491. Mutations that disrupt Gbetagamma interactions block PTH signaling via phospholipase Cbeta/[Ca2+]i and MAPK and markedly reduce signaling via adenylyl cyclase/cAMP. Herein, we define a domain on the PTH1R that is capable of binding G protein heterotrimeric complexes via direct Gbetagamma interactions.     

Modular design of Gbeta as the basis for reversible specificity in effector stimulation.

The G protein Gbetagamma subunit complex stimulates effectors by direct interactions utilizing extensive Gbeta regions over the surface of its propeller structure that faces the Galpha subunit. Our previous experiments have shown the resolved functions of signal transfer and general binding for Gbeta regions involved in stimulation of the effector phospholipase C-beta2, PLC-beta2, within the region Gbeta-(86-135), which comprises three beta strands arranged in a structurally contiguous fashion (Buck, E., Li, J., Chen, Y., Weng, G., Sacarlata, S., and Iyengar, R. (1999) Science 283, 1332-1335). This raises an important question as to why mutagenesis studies indicate that an extensive set of sites all over the Gbeta propeller structure and outside the 86-135 region are involved in Gbeta regulation of PLC-beta2. Using peptides to define functions of these Gbeta regions, we find that Gbeta signaling to PLC-beta2 relies on a collection of modular signal transfer and general binding units, each with lower apparent affinity relative to Gbetagamma-PLC interactions. Gbeta-(42-54) functions as a signal transfer region, Gbeta-(228-249) and Gbeta-(321-340) function in general binding, and Gbeta-(64-84) and Gbeta-(300-313) seem to play a structural role rather than a direct contact with the effector. A substitution within the Gbeta-(42-54) signal transfer region that increases the K(act) of this peptide for PLC-beta2 is accompanied by an increase in the observed maximal extent of signal transfer. We conclude that the lower K(act) for individual signal transfer regions may result in a decrease in the maximal effect of signal transfer. The spatial resolution of the signal transfer and general binding regions over a wide surface of Gbeta allow geometrical constraints to achieve specificity even with relatively low affinity interactions.     

Role of dynamic interactions in effective signal transfer for Gbeta stimulation of phospholipase C-beta 2

Heterotrimeric G protein subunits regulate their effectors by protein-protein interactions. The regions involved in these direct interactions have either signal transfer or general binding functions (Buck, E., Li, J., Chen, Y., Weng, G., Scarlata, S., and Iyengar, R. (1999) Science 283, 1332-1335). Although key determinants of signal transfer regions for G protein subunits have been identified, the mechanisms of signal transfer are not fully understood. We have used a combinatorial peptide approach to analyze one Gbeta region, Gbeta86-105, involved in signal transfer to the effector phospholipase C (PLC)-beta2 to gain a more mechanistic understanding of Gbeta/PLC-beta2 signaling. Binding and functional studies with the combinatorial peptides on interaction with and stimulation/inhibition of phospholipase Cbeta2 indicate that binding affinity can be resolved from EC(50) for functional effects, such that peptides that have wild type binding affinities have 15- to 20-fold lower EC(50) values. Although more potent, these peptides display a much lower extent of maximal stimulation. These peptides synergize with Gbetagamma or peptides encoding the second Gbeta42-54 signal transfer region in maximally stimulating phospholipase C-beta2. Other combinatorial peptides from the Gbeta86-105 region that bind to PLC-beta2 by themselves submaximally stimulate and extensively inhibit Gbetagamma stimulation of PLC-beta2. The intrinsic stimulation function can be attributed to Arg-96 and Ser-97, the synergy function to Trp-99, and the binding affinity to Thr-87, Val-90, Pro-94, Arg-96, Ser-97, and Val-100. These results indicate that, even within signal transfer regions, residues involved in binding can be resolved from those involved in signal transfer and that signal transfer is likely to be achieved through dynamic rather than steady-state interactions.     

The interaction of phospholipase C-beta3 with Shank2 regulates mGluR-mediated calcium signal

Phospholipase C-beta isozymes that are activated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins carry a PSD-95/Dlg/ZO-1 (PDZ) domain binding motif at their C terminus. Through interactions with PDZ domains, this motif may endow the PLC-beta isozyme with specific roles in GPCR signaling events that occur in compartmentalized regions of the plasma membrane. In this study, we identified the interaction of PLC-beta3 with Shank2, a PDZ domain-containing multimodular scaffold in the postsynaptic density (PSD). The C terminus of PLC-beta3, but not other PLC-beta isotypes, specifically interacts with the PDZ domain of Shank2. Homer 1b, a Shank-interacting protein that is linked to group I metabotropic glutamate receptors and IP3 receptors, forms a multiple complex with Shank2 and PLC-beta3. Importantly, microinjection of a synthetic peptide specifically mimicking the C terminus of PLC-beta3 markedly reduces the mGluR-mediated intracellular calcium response. These results demonstrate that Shank2 brings PLC-beta3 closer to Homer 1b and constitutes an efficient mGluR-coupled signaling pathway in the PSD region of neuronal synapses.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Galpha and Gbetagamma subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Galpha and Gbetagamma) following agonist-induced GPCR (alpha(2A)-adrenergic receptor; alpha(2A)-AR) activation in a cell-free assay system. alpha(2A)-AR membranes were reconstituted with the G-proteins (+/-hexahistidine-tagged) Galpha(i1) and Gbeta1gamma2 and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [35S]GTPgammaS. In the presence of Ni(2+)-coated agarose beads, the activated his-tagged Galpha(i1)his-[35S]GTPgammaS complex was captured on the Ni(2+)-presenting surface. When his-tagged Gbeta1gamma2 (Gbeta1gamma2his) was used with Galpha(i1), the [35S]GTPgammaS-bound Galpha(i1) was not present on the Ni(2+)-coated beads, but rather, it was separated from the beta1gamma2(his)-beads, demonstrating receptor-induced dissociation of Galpha and Gbetagamma subunits. Treatment of the reconstituted alpha(2A)-AR membranes containing Gbeta1gamma2his:Galpha(i1) with imidazole confirmed the specificity for the Ni2+:G-protein surface dissociation of Galpha(i1) from Gbeta1gamma2his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Differential sensitivity of P-Rex1 to isoforms of G protein betagamma dimers

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gbetagamma subunits of heterotrimeric G proteins. We examined which of the multiple G protein alpha and betagamma subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF(-)(4) activated G(s),G(i),G(q),G(12), or G(13) alpha subunits were unable to activate P-Rex1. Gbetagamma dimers containing Gbeta(1-4) complexed with gamma(2) stimulated P-Rex1 activity with EC(50) values ranging from 10 to 20 nm. Gbeta(5)gamma(2) was not able to stimulate P-Rex1 GEF activity. Dimers containing the beta(1) subunit complexed with a panel of different Ggamma subunits varied in their ability to stimulate P-Rex1. The beta(1)gamma(3), beta(1)gamma(7), beta(1)gamma(10), and beta(1)gamma(13HA) dimers all activated P-Rex1 with EC(50) values ranging from 20 to 38 nm. Dimers composed of beta(1)gamma(12) had lower EC(50) values (approximately 112 nm). The farnesylated gamma(11) subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (beta(1)gamma(11)) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gbetagamma dimer released by receptor activation may differentially activate P-Rex1.