Recent advances in molecular biology have shown GTPases and phosphoproteins to be the paramount molecular switches utilized intracellularly in biological systems. The origins of the GTPase switch appear to be almost as ancient as life itself, and through evolution nature has adapted this switch to a variety of purposes. In this two-volume work a broad survey of the major classes of GTPases is presented. The role of GTPases in ensuring accuracy during protein translation, a new look at the trimeric G-protein cycle, the molecular function of ARF in vesicle coating, the emerging role of the dynamin family in vesicle transfer, GTPases which activate GTPases during nascent protein translocation, and the many roles of ras-related proteins in growth, cytoskeletal polymerization, and vesicle transfer, are all described in 80 chapters by the leading authorities in their fields. Both detailed knowledge of specific systems or proteins and general principles of structure and function are offered. Much of this information has never been published before. At the rate the extended family of GTPases is growing it becomes increasingly unlikely that we will again get it to sit for a group portrait such as this. Therefore, the volume has the chance to become the reference work for GTPases.

Persistent directional movement of neutrophils in shallow chemotactic gradients raises the possibility that cells can increase their sensitivity to the chemotactic signal at the front, relative to the back. Redistribution of chemoattractant receptors to the anterior pole of a polarized neutrophil could impose asymmetric sensitivity by increasing the relative strength of detected signals at the cell's leading edge. Previous experiments have produced contradictory observations with respect to receptor location in moving neutrophils. To visualize a chemoattractant receptor directly during chemotaxis, we expressed a green fluorescent protein (GFP)-tagged receptor for a complement component, C5a, in a leukemia cell line, PLB-985. Differentiated PLB-985 cells, like neutrophils, adhere, spread, and polarize in response to a uniform concentration of chemoattractant, and orient and crawl toward a micropipette containing chemoattractant. Recorded in living cells, fluorescence of the tagged receptor, C5aR-GFP, shows no apparent increase anywhere on the plasma membrane of polarized and moving cells, even at the leading edge. During chemotaxis, however, some cells do exhibit increased amounts of highly folded plasma membrane at the leading edge, as detected by a fluorescent probe for membrane lipids; this is accompanied by an apparent increase of C5aR-GFP fluorescence, which is directly proportional to the accumulation of plasma membrane. Thus neutrophils do not actively concentrate chemoattractant receptors at the leading edge during chemotaxis, although asymmetrical distribution of membrane may enrich receptor number, relative to adjacent cytoplasmic volume, at the anterior pole of some polarized cells. This enrichment could help to maintain persistent migration in a shallow gradient of chemoattractant.

Disease-causing mutations often reveal key pathways of physiologic regulation and their underlying molecular mechanisms. Mutations in the trimeric guanine nucleotide-binding proteins (G proteins), which relay signals initiated by photons, odorants, and a host of hormones and neurotransmitters, cause many diseases. For the most part, the diseases are confined to a set of fascinating but rare endocrine disorders (Table 1).1 A recent study suggests that mutations in G proteins can also lead to essential hypertension.2 If this study is correct, hypertension may be one of several common disorders caused by defects in this ubiquitous family of signaling molecules. This review focuses . . .

Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the G(i) subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein beta gamma subunits and can be triggered by agonists for receptors coupled to G(i) but not by agonists for receptors coupled to two other G proteins, G(s) and G(q). Because activation of any G protein presumably releases free G beta gamma, we tested the hypothesis that chemotaxis also requires activated alpha subunits (G alpha(i)) of G(i) proteins. HEK293 cells were stably cotransfected with the G(i)-coupled receptor for interleukin-8, CXCR1, and with a chimeric G alpha G alpha(qz5) which resembles G alpha(i) in susceptibility to activation by G(i)-coupled receptors but cannot regulate the G alpha(i) effector, adenylyl cyclase, These cells, unlike cells expressing CXCR1 alone, migrated toward interleukin-8 even after treatment with pertussis toxin, which prevents activation of endogenous G alpha(i) but not that of G alpha(qz5). We infer that chemotaxis does not require activation of G alpha(i). Because chemotaxis is mediated by G beta gamma subunits released when G(i)-coupled receptors activate G alpha(qz5) but not when G(q)- or G(s)-coupled receptors activate their respective G proteins, we propose that G(i)-coupled receptors transmit a necessary chemotactic signal that is independent of G alpha(i).

The subcellular location of a signaling protein determines its ability to transmit messages accurately and efficiently. Three different lipid modifications tether heterotrimeric G proteins to membranes: alpha subunits are myristoylated and/or palmitoylated, and gamma subunits are prenylated, In a previous study, we examined the role of lipid modifications in maintaining the membrane attachment of a G protein alpha subunit, alpha(z), which is myristoylated and palmitoylated (Morales, J., Fishburn, C. S., Wilson, P. T., and Bourne, H. R. (1998) Mel. Biol. Cell 9, 1-14), Now we extend this analysis by characterizing the mechanisms that target newly synthesized alpha(z) to the plasma membrane (PM) and analyze the role of lipid modifications in this process. In comparison with newly synthesized alpha(s), which is palmitoylated but not myristoylated, alpha(z) moves more rapidly to the membrane fraction following synthesis in the cytosol, Newly synthesized alpha(z) associates randomly with cellular membranes, but with time accumulates at the PM. Palmitoylated alpha(z) is present only in PM-enriched fractions, whereas a nonpalmitoylated mutant of alpha(z) (alpha(z) C3A) associates less stably with the PM than does wild-type alpha(z). Expression of a C-terminal fragment of the beta-adrenoreceptor kinase, which sequesters free beta gamma, impairs association of both alpha(z) and alpha(z) C3A with the PM, suggesting that the alpha subunit must bind beta gamma in order to localize at the PM. Based on these findings, we propose a model in which, following synthesis on soluble ribosomes, myristoylated a, associates randomly and reversibly with membranes; upon association with the PM, alpha(z) binds beta gamma, which promotes its palmitoylation, thus securing it in the proper place for transmitting the hormonal signal.

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by a subunits of the G(i) family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits G(i)-mediated hormonal signals probably by acting as GTPase-activating proteins for G alpha(i) subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the G(q/11) family, we compared activation of mitogen-activated protein kinase (MAPK) by a G(q/11)-coupled receptor, the bombesin receptor (BR), and a G(i)-coupled receptor, the D-2 dopamine receptor, transiently co expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples G(i) from receptors, blocked MAPK activation by the D-2 dopamine receptor but not by the BR. Go-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists, RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of G(q/11), phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alpha(q), probably by binding to alpha(q).GDP.AlF4-. These results demonstrate that RGS4 can regulate G(q/11)-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.

Many chemoattractants cause chemotaxis of leukocytes by stimulating a structurally distinct class of G protein-coupled receptors. To identify receptor functions required for chemotaxis, we studied chemotaxis in HEK293 cells transfected with receptors for nonchemokine ligands or for interleukin 8 (IL-8), a classical chemokine. In gradients of the appropriate agonist, three nonchemokine Gi-coupled receptors (the D2 dopamine receptor and opioid mu and delta receptors) mediated chemotaxis; the beta2-adrenoreceptor and the M3-muscarinic receptor, which couple respectively to Gs and Gq, did not mediate chemotaxis. A mutation deleting 31 C-terminal amino acids from the IL-8 receptor type B quantitatively impaired chemotaxis and agonist-induced receptor internalization, but not inhibition of adenylyl cyclase or stimulation of mitogen-activated protein kinase. To probe the possible relation between receptor internalization and chemotaxis, we used two agonists of the mu-opioid receptor. Morphine and etorphine elicited quantitatively similar chemotaxis, but only etorphine induced receptor internalization. Overexpression of two betagamma sequestering proteins (betaARK-ct and alphat) prevented IL-8 receptor type B-mediated chemotaxis but did not affect inhibition of adenylyl cyclase by IL-8. We conclude that: (i) Nonchemokine Gi-coupled receptors can mediate chemotaxis. (ii) Gi activation is necessary but probably not sufficient for chemotaxis. (iii) Chemotaxis does not require receptor internalization. (iv) Chemotaxis requires the release of free betagamma subunits.

One of the first to be discovered and still one of the most important, cyclic AMP is a second messenger inside cells, carrying information from outside signals to change processes inside. As described by Bourne in his Perspective and reported by Tesmer et al. and Sunahara et al., adenylyl cyclase, the enzyme that catalyzes this process, has now been crystallized with the molecule that turns it on, the G protein G, yielding a first glimpse into the molecular details that underly this critical biochemical reaction.

X-ray or NMR structures of proteins are often derived without their ligands, and even when the structure of a full complex is available, the area of contact that is functionally and energetically significant may be a specialized subset of the geometric interface deduced from the spatial proximity between ligands. Thus, even after a structure is solved, it remains a major theoretical and experimantal goal to localize protein functional interfaces and understand the role of their constituent residues. The evolutionary trace method is a systematic, transparent and novel predictive technique that identifies active sites and functional interfaces in proteins with known structure. It is based on the extraction of functionally important residues from sequence conservation patterns in homologous proteins, and on their mapping onto the protein surface to generate clusters identifying functional interfaces. The SH2 and SH3 modular signaling domains and the DNA binding domain of the nuclear hormone receptors provide tests for the accuracy and validity of our method. In each case, the evolutionary trace deliineates the functional epitope and identifies residues critical to the binding specificity. Based on mutational evolutionary analysis and on the structural homology of protein families, this simple and versatile approach should help focus site-directed mutagenesis studies of structure-function relationships in macromolecules, as well as studies of specificity in molecular recognitionl. More generally, it provedes an evolutionary perspective for judging the functional or structural role of each residue in a protein structure.

Many growth factors and agonists for G protein-coupled receptors activate mitogen-activated protein (MAP) kinase pathways, including the extracellular signal-regulated kinase (ERK) pathway and the c-Jun kinase (JNK) pathway. Transient transfection of dominant negative and constitutively active pathway components in COS-7 cells shows that two G protein subunits, Galpha12 and Galpha13, inhibit the ERK pathway and stimulate the JNK pathway. Constitutively active (GTPase-deficient) Galpha12 and Galpha13 both inhibit ERK pathway activation by epidermal growth factor. A Galpha13/alphaz chimera, which responds to stimulation by Gi-coupled receptors, mediates inhibition of ERK via such a receptor, the dopamine-2 receptor. In addition, expression of a dominant negative mutant of the GTPase, Cdc42, blocks activation of the JNK pathway by Galpha12 and Galpha13 but does not alter inhibition of ERK activation by the same Galpha proteins; conversely, mutationally activated Cdc42 stimulates the JNK pathway but has no effect on the ERK pathway. Our results show that different mechanisms mediate two effects of Galpha12 and Galpha13: the ERK pathway inhibition is mediated at the level of MAP kinase kinase in a Ras- and Raf-independent fashion, whereas the JNK pathway stimulation is mediated by Cdc42.

Pseudohypoparathyroidism, type Ia (PHP-Ia), is a dominantly inherited endocrine disorder characterized by resistance to hormones that act by stimulating adenylyl cyclase. It is caused by inheritance of an autosomal mutation that inactivates the alpha subunit (alphas) of Gs, the stimulatory regulator of adenylyl cyclase. In three members of a family, the PHP-Ia phenotype is associated with a mutation (R231H) that substitutes histidine for an arginine at position 231 in alphas. We assessed signaling function of alphas-WT versus alphas-R231H transiently transfected in HEK293 cells. Hormone receptor-dependent stimulation of cAMP accumulation in cells expressing alphas-R231H is reduced by approximately 75% in comparison to cAMP accumulation in cells expressing alphas-WT. A second mutation, alphas-R201C, inhibits the GTPase turnoff reaction of alphas, thus producing receptor-independent stimulation of cAMP accumulation. The double mutant, alphas-R231H/R201C, stimulates cAMP accumulation almost as well (approximately 80%) as does alphas-R201C itself, indicating that the R231H mutation selectively impairs receptor-dependent signaling. In three-dimensional structures of G protein heterotrimers, Arg-231 is located in a region, switch 2, that is thought to interact with the betagamma subunit rather than with the hormone receptor. Thus, the R231H phenotype suggests that switch 2 (perhaps in concert with betagamma) mediates G protein activation by receptors at a site distant from the receptor-G protein contact surface.

We have examined the subcellular distribution of alpha s, the alpha subunit of the heterotrimeric G protein Gs, by using immunofluorescence microscopy. In transiently transfected HEK293 cells, wild-type alpha s localizes to the plasma membrane. However, a mutationally activated alpha s (alpha sR201C) is diffusely distributed throughout the cytoplasm. Similarly, cholera toxin activation of alpha s causes it to redistribute from the plasma membrane to cytoplasm in stably transfected cells. In HEK293 cells stably transfected with alpha s and the beta 2-adrenergic receptor (beta-AR), stimulation of the beta-AR by the agonist isoproterenol also causes a translocation of alpha s from the plasma membrane to cytoplasm. Replacing the agonist with antagonist allows alpha s to return to the plasma membrane, demonstrating the reversibility of alpha s translocation. Receptor-activated alpha s does not colocalize with internalized beta-AR at endosomes. Incubation of cells in hypertonic sucrose to inhibit clathrin-coated pit-mediated endocytosis of agonist-activated beta-AR failed to block agonist-stimulated redistribution of alpha s. These findings demonstrate that activated alpha s reversibly undergoes a translocation from the plasma membrane to cytoplasm and begin to address the relationship between regulated trafficking of a seven-transmembrane receptor and its cognate G protein.

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.

The carboxyl terminus of the G protein alpha subunit is a key determinant of the fidelity of receptor activation. We have previously shown that the Gq alpha subunit (alpha q) can be made to respond to alpha i-coupled receptors by replacing its carboxyl terminus with the corresponding alpha i2, alpha o, alpha z residues. We now extend these findings in three ways: 1) carboxyl-terminal mutations of alpha q/alpha i chimeras show that the critical amino acids are in the -3 and -4 positions, 2) exchange of carboxyl termini between alpha q and alpha z allows activation by receptors appropriate to the carboxyl-terminal residues, and 3) we identify receptors that either do or do not activate the expected carboxyl-terminal chimeras (alpha q/alpha i, alpha q/alpha s, alpha s/alpha q). Replacement of the five carboxyl-terminal amino acids of alpha q with the alpha s sequence permitted an alpha s-coupled receptor (the V2 vasopressin receptor but not the beta 2-adrenergic receptor) to stimulate phospholipase C. Replacement of the five carboxyl-terminal amino acids of alpha z with residues of alpha q permitted certain alpha q-coupled receptors (bombesin and V1a vasopressin receptors but not the oxytocin receptor) to stimulate adenylyl cyclase. Thus, the relative importance of the G alpha carboxyl terminus in permitting coupling to a new receptor depends on the receptor with which it is paired. These studies refine our understanding and provide new tools with which to study the fidelity of receptor/G alpha activation.

As the first step in an investigation of roles played by fatty acylation of G protein alpha chains in membrane targeting and signal transmission, we inserted monoclonal antibody epitopes, hemagglutinin (HA) or Glu-Glu (EE), at two internal sites in three alpha subunits. At site I, only HA-tagged alpha q and alpha z functioned normally. alpha s, alpha q, and alpha z subunits tagged at site II with the EE epitope showed normal expression, membrane localization, and signaling activity. Using epitope-tagged alpha z, we investigated effects of mutations in sites for fatty acylation. Mutational substitution of Ala for Gly2 (G2A) prevented incorporation of myristate and decreased but did not abolish incorporation of palmitate. Substitution of Ala for Cys3 (C3A) prevented incorporation of palmitate but had no effect on incorporation of myristate. Substitution of Ala for both Gly2 and Cys3 (G2AC3A) prevented incorporation of both myristate and palmitate. All three mutations substantially disrupted association of alpha z with the particulate fraction. Gz-mediated inhibition of adenylyl cyclase, triggered by activation of the D2-dopamine receptor, was, respectively, abolished (G2AC3A), impaired (G2A), and enhanced (C3A). Constitutive inhibition of adenylyl cyclase by alpha z was unchanged (G2AC3A), strongly diminished (G2A), or strongly enhanced (C3A). A nonacylated, mutationally activated alpha z mutant inhibited adenylyl cyclase, although less potently than normally acylated, mutationally activated alpha z. From these findings we conclude: (a) fatty acylations of alpha z increase its association with membranes; (b) myristoylation is not required for palmitoylation of alpha z or for its productive interactions with adenylyl cyclase; (c) palmitoylation is not required for, but may instead inhibit, signaling by alpha z.