Identification of receptor contact site involved in receptor-G protein coupling

The mammalian G proteins transduce information from extracellular signals, including neurotransmitters, hormones and sensory stimuli, into regulation of effector enzymes or ion channels within cells. Triggered by appropriate extracellular signals, receptor proteins specifically activate members of the G protein family by catalysing replacement of GDP by GTP at the guanine nucleotide binding site. Like the receptor proteins, the heterotrimeric G proteins exhibit impressive structural similarities, suggesting that all receptor-G protein interactions use homologous structural elements and a single molecular mechanism. Topologically equivalent portions of each G protein may therefore interact with the appropriate receptor. We recently predicted the secondary structure of a composite G protein alpha-chain and proposed that a predicted amphipathic alpha-helix at the extreme carboxy-terminus of the polypeptide directly contacts receptors. This proposal has now been confirmed by sequencing complementary DNAs of the gene that encodes the alpha-chain (alpha s) of the stimulatory regulator (Gs) of adenylyl cyclase in wild-type cells and in a mutant mouse S49 lymphoma cell line, unc, in which Gs cannot be activated by hormone receptors. The sequences reveal a point mutation in the unc gene that substitutes a proline residue for an arginine near the carboxy-terminus of the alpha s-polypeptide. Expression of recombinant alpha s-unc in genetically alpha s-deficient S49 cells reproduces the unc phenotype.     

Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits.

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits. In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively. By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways. Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma. This effect of beta gamma requires costimulation by alpha s-GTP. This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active. We now confirm this prediction. Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis. Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation. These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.     

Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding

Transducin, composed of subunits T alpha, T beta and T gamma, is a member of a heterotrimeric G-protein family, and transduces the light signal in visual cells. We have recently found that bovine T beta gamma can be separated into two components. T beta gamma-1 and T beta gamma-2, each of which has its own gamma-subunit, T gamma-1 and T gamma-2, respectively. T beta gamma-2 enhances the binding of GTP to T alpha in the presence of metarhodopsin II by about 30-fold compared with T beta gamma-1. Here we show that a farnesyl moiety is attached to a sulphur atom of the C-terminal cysteine of T gamma-2 (active form), a part of which is additionally methyl-esterified at the alpha-carboxyl group. In T gamma-1 (inactive form), however, such modifications are missing. Thus, the farnesyl moiety attached to the gamma-subunit is indispensable for the GTP-binding activity of transducin. This suggests that a similar modification may occur in the gamma-subunits of other heterotrimeric G proteins involved in biological signal transduction processes.     

Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C.

The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).     

Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature

Protein coats deform flat lipid membranes into buds and capture membrane proteins to form transport vesicles. The assembly/disassembly cycle of the COPI coat on Golgi membranes is coupled to the GTP/GDP cycle of the small G protein Arf1. At the heart of this coupling is the specific interaction of membrane-bound Arf1-GTP with coatomer, a complex of seven proteins that forms the building unit of the COPI coat. Although COPI coat disassembly requires the catalysis of GTP hydrolysis in Arf1 by a specific GTPase-activating protein (ArfGAP1), the precise timing of this reaction during COPI vesicle formation is not known. Using time-resolved assays for COPI dynamics on liposomes of controlled size, we show that the rate of ArfGAP1-catalysed GTP hydrolysis in Arf1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle. This leads to a model for COPI dynamics in which GTP hydrolysis in Arf1 is organized temporally and spatially according to the changes in lipid packing induced by the coat.     

Subsecond deactivation of transducin by endogenous GTP hydrolysis

The response of a retinal rod cell to a weak flash of light is mediated by a receptor/GTP-binding protein (rhodopsin/transducin) signal transduction system and terminates within a second. The T alpha subunit of transducin (composed of subunits T alpha, T beta and T gamma) is triggered by photoexcited rhodopsin (R*) to release GDP and bind GTP. The binding of GTP causes release of the T alpha unit from T beta gamma and allows it to modulate the activity of an enzyme that generates a second messenger. Termination of the response requires the hydrolysis of the GTP by intrinsic GTPase. As with other G proteins, the GTPase activity of transducin seems to be slow. Reported in vitro turnover rates of a few molecules of GTP hydrolysed per molecule of transducin per minute imply a T alpha-GTP deactivation time of many seconds. But this time might be only a small fraction of that of the GTPase cycle. We have now used time-resolved microcalorimetry in bovine rod outer segments (ROS) to monitor the heat release due to the hydrolysis of GTP by a transducin population that had been quickly activated by flash illumination of rhodopsin. The enthalpy of GTP hydrolysis is released within 1 s at 23 degrees C. This deactivation time seems to be independent of any diffusible factor in the preparation and concurs with the termination kinetics of the rod's response. Thereafter, transducin seems unable to reload GTP for many seconds. This refractory 'resetting' time may account for the low steady-state GTPase rates in vitro.     

Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK.

The proteins Cdc42 and Rac are members of the Rho family of small GTPases (G proteins), which control signal-transduction pathways that lead to rearrangements of the cell cytoskeleton, cell differentiation and cell proliferation. They do so by binding to downstream effector proteins. Some of these, known as CRIB (for Cdc42/Rac interactive-binding) proteins, bind to both Cdc42 and Rac, such as the PAK1-3 serine/threonine kinases, whereas others are specific for Cdc42, such as the ACK tyrosine kinases and the Wiscott-Aldrich-syndrome proteins (WASPs). The effector loop of Cdc42 and Rac (comprising residues 30-40, also called switch I), is one of two regions which change conformation on exchange of GDP for GTP. This region is almost identical in Cdc42 and Racs, indicating that it does not determine the specificity of these G proteins. Here we report the solution structure of the complex of Cdc42 with the GTPase-binding domain ofACK. Both proteins undergo significant conformational changes on binding, to form a new type of G-protein/effector complex. The interaction extends the beta-sheet in Cdc42 by binding an extended strand from ACK, as seen in Ras/effector interactions, but it also involves other regions of the G protein that are important for determining the specificity of effector binding.     

Different beta-subunits determine G-protein interaction with transmembrane receptors.

Regulatory GTP-binding proteins (G proteins) are membrane-attached heterotrimers (alpha, beta, gamma) that mediate cellular responses to a wide variety of extracellular stimuli. They undergo a cycle of guanine-nucleotide exchange and GTP hydrolysis, during which they dissociate into alpha-subunit and beta gamma complex. The roles of G-protein alpha-subunits in these processes and for the specificity of signal transduction are largely established; the beta- and gamma-subunits are essential for receptor-induced G-protein activation and seem to be less diverse and less specific. Although the complementary DNAs for several beta-subunits have been cloned, isolated subunits have only been studied as beta gamma complexes. Functional differences have been ascribed to the gamma-subunit on the basis of extensive sequence similarity among beta-subunits and apparent heterogeneity in gamma-subunit sequences. Beta gamma complexes can interact directly or indirectly with different effectors. They seem to be interchangeable in their interaction with pertussis toxin-sensitive alpha-subunits, so we tested this by microinjecting antisense oligonucleotides into nuclei of a rat pituitary cell line to suppress the synthesis of individual beta-subunits selectively. Here we show that two out of four subtypes of beta-subunits tested (beta 1 and beta 3) are selectively involved in the signal transduction cascades from muscarinic M4 (ref. 4) and somatostatin receptors, respectively, to voltage-dependent Ca2+ channels.     

How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP

Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells. Interferon-gamma-inducible human guanylate binding protein 1 (hGBP1) belongs to the family of dynamin-related large GTP-binding proteins, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the beta-phosphates in the gamma-phosphate-binding site.     

Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 A

A multitude of heptahelical receptors use heterotrimeric G proteins to transduce signals to specific effector target molecules. The G protein transducin, Gt, couples photon-activated rhodopsin with the effector cyclic GMP phosophodiesterase (PDE) in the vertebrate phototransduction cascade. The interactions of the Gt alpha-subunit (alpha(t)) with the inhibitory PDE gamma-subunit (PDEgamma) are central to effector activation, and also enhance visual recovery in cooperation with the GTPase-activating protein regulator of G-protein signalling (RGS)-9 (refs 1-3). Here we describe the crystal structure at 2.0 A of rod transducin alpha x GDP x AlF4- in complex with the effector molecule PDEgamma and the GTPase-activating protein RGS9. In addition, we present the independently solved crystal structures of the RGS9 RGS domain both alone and in complex with alpha(t/i1) x GDP x AlF4-. These structures reveal insights into effector activation, synergistic GTPase acceleration, RGS9 specificity and RGS activity. Effector binding to a nucleotide-dependent site on alpha(t) sequesters PDEgamma residues implicated in PDE inhibition, and potentiates recruitment of RGS9 for hydrolytic transition state stabilization and concomitant signal termination.     

An immunological role for the CD8 beta-chain.

Mature T cells can be functionally divided into two categories distinguished by surface expression of either CD4 or CD8, which in turn corresponds to restriction by and binding to class II or class I major histocompatibility complex proteins, respectively. CD8 can be expressed as a homodimer of the alpha-chain, or as a heterodimer of alpha- and beta-chains on human and mouse T cells, although most peripheral T cells seem to express CD8 alpha beta heterodimers exclusively (reviewed in ref. 9). Functional characterization of CD8 has focused primarily on the effect of the alpha-chain, which enhances or reconstitutes T-cell responses in homodimeric form and may play a specific role in thymic selection. In contrast, no role has been ascribed to CD8 beta or alpha beta heterodimers specifically. Here we show that CD8 alpha beta transfectants produce more interleukin-2 than CD8 alpha transfectants in response to specific stimuli. Increased interleukin-2 is also observed in cells expressing hybrid CD8 beta-alpha molecules (extracellular CD8 beta plus CD8 alpha transmembrane and cytoplasmic regions) on their surface. These results indicate that external portions of CD8 beta could be critical and that they may act independently of CD8 alpha in mediating their augmentation effect.     

Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins.

Many hormones, neurotransmitters and growth factors, on binding to G protein-coupled receptors or receptors possessing tyrosine kinase activity, increase intracellular levels of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This is due to activation of phosphoinositide-specific phospholipase(s) C (PLC), the isozymes of which are classified into groups, alpha, beta, gamma and delta. The beta, gamma and delta groups themselves contain PLC isozymes which have both common and unique structural domains. Only the gamma 1 isozyme has been implicated in a signal transduction mechanism. This involves association with, and tyrosine phosphorylation by, the ligand-bound epidermal growth factor and platelet-derived growth factor receptors, probably by means of the PLC-gamma 1-specific src homology (SH2) domain. Because EGF receptor-mediated tyrosine phosphorylation of PLC-gamma 1 stimulates catalytic activity in vitro and G proteins have been implicated in the activation of PLC, we investigated which PLC isozymes are subject to G protein regulation. We have purified an activated G protein alpha subunit that stimulates partially purified phospholipase C and now report that this G protein specifically activates the beta 1 isozyme, but not the gamma 1 and delta 1 isozymes of phospholipase C. We also show that this protein is related to the Gq class of G protein alpha subunits.     

Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein.

The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle. Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif. One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin. Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema. Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP. An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42. A carboxy-terminal beta-hairpin and alpha-helix pack against switch II. The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42. Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases. Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors.     

The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product

About 30% of human tumours contain a mutation in one of the three ras genes leading to the production of p21ras oncoproteins that are thought to make a major contribution to the transformed phenotype of the tumour. The biochemical mode of action of the ras proteins is unknown but as they bind GTP and GDP and have an intrinsic GTPase activity, they may function like regulatory G proteins and control cell proliferation by regulating signal transduction pathways at the plasma membrane. It is assumed that an external signal is detected by a membrane molecule (or detector) that stimulates the conversion of p21.GDP to p21.GTP which then interacts with a target molecule (or effector) to generate an internal signal. Recently a cytoplasmic protein, GAP, has been identified that interacts with the ras proteins, dramatically increasing the GTPase activity of normal p21 but not of the oncoproteins. We report here that GAP appears to interact with p21ras at a site previously identified as the 'effector' site, strongly implicating GAP as the biological target for regulation by p21.     

Lipid modification at the N terminus of photoreceptor G-protein alpha-subunit.

Myristate is a fatty acid (fourteen-carbon chain with no double bonds, C14:0) linked to the amino-terminal glycine of several proteins, including alpha-subunits of heterotrimeric (alpha/beta gamma) G proteins. We report here a novel modification at the N terminus of the alpha-subunit of the photoreceptor G protein transducin, T alpha, with heterogeneous fatty acids composed of laurate (C12:0), unsaturated C14:2 and C14:1 fatty acids, and a small amount (approximately 5%) of myristate. Both the GTPase activity of T alpha/T beta gamma and the T beta gamma-dependent ADP-ribosylation of T alpha catalysed by pertussis toxin were inhibited by the lauroylated and myristoylated N-terminal peptide of T alpha. The myristoylated peptide gave 50% inhibition at a 3.5 to approximately 4.5-fold lower concentration than the lauroylated peptide in each assay, indicating that the strength of the interaction between T alpha and T beta gamma is altered by heterogeneous fatty acids linked to T alpha. This suggests that a looser subunit interaction in transducin which is due to an abundance of N-linked fatty acids other than myristate would favour the rapid turnover and catalysis essential for the visual excitation in photoreceptor cells.     

A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion.

The union of sperm and egg is a special membrane fusion event that gives a signal to begin development. We have hypothesized that proteins mediating cell-cell fusion events resemble viral fusion proteins and have shown that PH-30, a sperm surface protein involved in sperm-egg fusion, shares biochemical characteristics with viral fusion proteins. We report here the complementary DNA and deduced amino-acid sequences of the mature alpha and beta subunits of PH-30. Both are type-I integral membrane glycoproteins. The alpha subunit contains a putative fusion peptide typical of viral fusion proteins and the beta subunit contains a domain related to a family of soluble integrin ligands found in snake venoms. Thus, the PH-30 alpha/beta complex resembles many viral fusion proteins in both its membrane topology and its predicted binding and fusion functions.     

Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.     

GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours

A subset of growth hormone-secreting human pituitary tumours carries somatic mutations that inhibit GTPase activity of a G protein alpha chain, alpha(s). The resulting activation of adenylyl cyclase bypasses the cells' normal requirement for trophic hormone. Amino acids substituted in the putative gsp oncogene identify a domain of G protein alpha-chains required for intrinsic ability to hydrolyse GTP. This domain may serve as a built-in counter-part of the separate GTPase-activating proteins required for GTP hydrolysis by small GTP-binding proteins such as p21ras.     

Antisense oligodeoxynucleotides to GS protein alpha-subunit sequence accelerate differentiation of fibroblasts to adipocytes.

Fully-differentiated mouse 3T3-L1 fibroblasts accumulate large amounts of lipid at 7-10 days after induction by insulin or by dexamethasone and a methyl xanthine. G proteins mediate transmembrane signalling from a diverse group of cell-surface receptors to effector units that include phospholipase C, adenylyl cyclase and ion channels. They are also targets of regulation themselves. 3T3-L1 fibroblasts display marked changes in levels of G protein when induced to differentiate to adipocytes. Here we show that cholera toxin, which ADP-ribosylates and activates the G protein subunit Gs alpha, blocks the induction of differentiation, whereas increasing intracellular cyclic AMP directly with the dibutyryl analogue or indirectly with pertussis toxin or forskolin does not affect differentiation. Oligodeoxynucleotides antisense to the sequence encoding Gs alpha accelerate differentiation markedly. The time course of adipogenesis declined from 7-10 days in controls to roughly 3 days in cultures treated with antisense-Gs alpha oligodeoxynucleotides, whereas oligodeoxynucleotides, antisense to Gi alpha 1, Gi alpha 3, and sense and missense to Gs alpha, had no such effect. Antisense-Gs alpha alone induced differentiation by day 7, indicating that Gs alpha activity modulates differentiation in 3T3-L1 cells, acting in a new role which is independent of increased intracellular cAMP.     

Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits.

Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.