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1.
Article in English | MEDLINE | ID: mdl-17703575

ABSTRACT

From the point of view of a participant observer, I tell the discovery stories of trimeric G-proteins and GPCRs, beginning in the 1970s. As in most such stories, formidable obstacles, confusion, and mistakes make eventual triumphs even more exciting. Because these pivotally important signaling molecules were discovered before the recombinant DNA revolution, today's well-trained molecular biologist may find it amazing that we learned anything at all.


Subject(s)
GTP-Binding Proteins/physiology , Receptors, G-Protein-Coupled/physiology , GTP-Binding Proteins/history , History, 20th Century , Humans , Receptors, G-Protein-Coupled/history
2.
Trends Pharmacol Sci ; 22(11): 587-93, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11698103

ABSTRACT

G-protein-coupled receptors (GPCRs) are a large family of seven-transmembrane-helix proteins that mediate responses to hormones, neurotransmitters and, in the case of rhodopsin, photons. The recent determination of the structure of rhodopsin at atomic resolution opens avenues to a deeper understanding of GPCR activation and transmembrane signaling. Data from previous crosslinking, spin labeling and scanning accessibility experiments on rhodopsin have been mapped onto the high-resolution structure. These data correlate well and are consistent with the structure, and suggest that activation by light opens a cleft at the cytoplasmic end of the seven-helix bundle of rhodopsin. Furthermore, lessons learned from rhodopsin might also apply to other members of this essential family of receptors. (For an animation of the crystal structure of rhodopsin see http://archive.bmn.com/supp/tips/tips2211a.html)


Subject(s)
Rhodopsin/chemistry , Rhodopsin/physiology , Animals , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/physiology , Humans , Models, Molecular , Protein Conformation
3.
Proc Natl Acad Sci U S A ; 98(11): 6150-5, 2001 May 22.
Article in English | MEDLINE | ID: mdl-11344266

ABSTRACT

How receptors catalyze exchange of GTP for GDP bound to the Galpha subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's betagamma heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Galpha. To test this possibility, we designed a mutant Galpha that would bind to betagamma in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of alpha(s), the alpha subunit of G(S), the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed beta(1) and gamma(2), this mutant (alpha(s)Delta), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed beta protein to interact normally with the lip of the nucleotide binding pocket of alpha(s)Delta. We substituted alanine for an aspartate in beta(1) that binds to a lysine (K206) in the lip of the alpha subunit's nucleotide binding pocket. Coexpressed with alpha(s)Delta and gamma(2), this mutant, beta(1)-D228A, elevated cAMP much less than did beta(1)-wild type; it did bind to alpha(s)Delta normally, however, as indicated by its unimpaired ability to target alpha(s)Delta to the plasma membrane. We conclude that betagamma can activate alpha(s) and that this effect probably involves both a tilt of betagamma relative to alpha(s) and interaction of beta with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.


Subject(s)
GTP-Binding Protein alpha Subunits, Gs/metabolism , Guanosine Diphosphate/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Line , Chlorocebus aethiops , GTP Phosphohydrolases , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/genetics , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/genetics , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis
4.
J Biol Chem ; 276(5): 3394-400, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11062244

ABSTRACT

Although agonists are thought to occupy binding pockets within the seven-helix core of serpentine receptors, the topography of these binding pockets and the conformational changes responsible for receptor activation are poorly understood. To identify the ligand binding pocket in the receptor for complement factor 5a (C5aR), we assessed binding affinities of hexapeptide ligands, each mutated at a single position, for seven mutant C5aRs, each mutated at a single position in the putative ligand binding site. In ChaW (an antagonist) and W5Cha (an agonist), the side chains at position 5 are tryptophan and cyclohexylalanine, respectively. Comparisons of binding affinities indicated that the hexapeptide residue at this position interacts with two C5aR residues, Ile-116 (helix III) and Val-286 (helix VII); in a C5aR model these two side chains point toward one another. Both the I116A and the V286A mutations markedly increased binding affinity of W5Cha but not that of ChaW. Moreover, ChaW, the antagonist hexapeptide, acted as a full agonist on the I116A mutant. These results argue that C5aR residues Ile-116 and Val-286 interact with the side chain at position 5 of the hexapeptide ligand to form an activation switch. Based on this and previous work, we present a docking model for the hexapeptide within the C5aR binding pocket. We propose that agonists induce a small change in the relative orientations of helices III and VII and that these helices work together to allow movement of helix VI away from the receptor core, thereby triggering G protein activation.


Subject(s)
Antigens, CD/chemistry , Receptors, Complement/chemistry , Signal Transduction/physiology , Alanine/genetics , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Binding Sites , COS Cells , Isoleucine/genetics , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Mutation , Peptides/chemistry , Protein Conformation , Receptor, Anaphylatoxin C5a , Receptors, Complement/genetics , Receptors, Complement/metabolism
5.
Trends Cell Biol ; 10(11): 466-73, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11050418

ABSTRACT

Morphologic polarity is necessary for the motility of mammalian cells. In leukocytes responding to a chemoattractant, this polarity is regulated by activities of small Rho guanosine triphosphatases (Rho GTPases) and the phosphoinositide 3-kinases (PI3Ks). Moreover, in neutrophils, lipid products of PI3Ks appear to regulate activation of Rho GTPases, are required for cell motility and accumulate asymmetrically to the plasma membrane at the leading edge of polarized cells. By spatially regulating Rho GTPases and organizing the leading edge of the cell, PI3Ks and their lipid products could play pivotal roles not only in establishing leukocyte polarity but also as compass molecules that tell the cell where to crawl.


Subject(s)
Chemotaxis, Leukocyte , Isoenzymes/metabolism , Leukocytes/physiology , Phosphatidylinositol 3-Kinases/metabolism , rho GTP-Binding Proteins/metabolism , Actins/metabolism , Animals , Cell Membrane/enzymology , Cell Polarity , Chemotactic Factors/pharmacology , Class Ib Phosphatidylinositol 3-Kinase , HL-60 Cells , Humans , Isoenzymes/genetics , Leukocytes/enzymology , Models, Biological , Phosphatidylinositol 3-Kinases/genetics , Signal Transduction
6.
Science ; 289(5480): 733-4, 2000 Aug 04.
Article in English | MEDLINE | ID: mdl-10950717

ABSTRACT

Members of the seven transmembrane receptor superfamily bind a remarkable variety of ligands, from neurotransmitters to odorants, and activate a spectacular array of G protein signaling molecules. These G-protein coupled receptors (GPCRs) are important in many cellular functions and so there has been great interest in elucidating how they transmit their signals to the interior of the cell after activation by ligand. As Bourne and Meng explain in their Perspective, the molecular movements of activated GPCRs are becoming clear now that the first crystal structure of a GPCR (rhodopsin, the light-trapping receptor found in the retina of the eye) has been reported (Palczweski et al.).


Subject(s)
Rhodopsin/chemistry , Binding Sites , Crystallography, X-Ray , Evolution, Molecular , Heterotrimeric GTP-Binding Proteins/metabolism , Ligands , Lipid Bilayers , Models, Molecular , Protein Structure, Secondary , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Retinaldehyde/metabolism , Rhodopsin/metabolism , Stereoisomerism , Vision, Ocular
7.
Science ; 287(5455): 1037-40, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10669415

ABSTRACT

Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant. Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.


Subject(s)
Bacterial Proteins , Cell Polarity , Chemotaxis, Leukocyte/physiology , Neutrophils/physiology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Signal Transduction , Actins/metabolism , Bacterial Toxins/pharmacology , Cell Membrane/enzymology , Chemotactic Factors/pharmacology , Chromones/pharmacology , Complement C5a/pharmacology , Cytoplasm/enzymology , Enzyme Inhibitors/pharmacology , HL-60 Cells , Humans , Insulin/pharmacology , Morpholines/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/enzymology , Neutrophils/ultrastructure , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt , Pseudopodia/enzymology , Receptors, Formyl Peptide , Recombinant Fusion Proteins/metabolism , rho GTP-Binding Proteins/antagonists & inhibitors , rho GTP-Binding Proteins/metabolism
8.
Proc Natl Acad Sci U S A ; 97(3): 1085-90, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10655488

ABSTRACT

To explore the relative roles of protein-binding partners vs. lipid modifications in controlling membrane targeting of a typical peripheral membrane protein, Galpha(z), we directed its binding partner, betagamma, to mislocalize on mitochondria. Mislocalized betagamma directed wild-type Galpha(z) and a palmitate-lacking Galpha(z) mutant to mitochondria but did not alter localization of a Galpha(z) mutant lacking both myristate and palmitate. Thus, in this paradigm, a protein-protein interaction controls targeting of a peripheral membrane protein to the proper compartment, whereas lipid modifications stabilize interactions of proteins with membranes and with other proteins.


Subject(s)
GTP-Binding Protein alpha Subunits , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Amino Acid Substitution , Animals , Biological Transport , CHO Cells , Cricetinae , Cricetulus , Heterotrimeric GTP-Binding Proteins/genetics , MAP Kinase Signaling System , Membrane Lipids/metabolism , Microscopy, Fluorescence , Mitochondria/metabolism , Mutagenesis, Site-Directed , Myristic Acid/metabolism , Palmitates/metabolism , Recombinant Fusion Proteins/metabolism , Transfection
9.
Curr Biol ; 9(23): R870-1, 1999 Dec 02.
Article in English | MEDLINE | ID: mdl-10607574
10.
Nat Cell Biol ; 1(2): 75-81, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10559877

ABSTRACT

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.


Subject(s)
Actins/blood , Chemotaxis, Leukocyte , Cytoskeletal Proteins , Neutrophils/cytology , Neutrophils/physiology , Actin-Related Protein 2 , Actins/chemistry , Actins/ultrastructure , Animals , Cell Membrane/physiology , Cell Membrane Permeability , Cell Polarity , Humans , Listeria monocytogenes/physiology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Rabbits , Signal Transduction
11.
J Biol Chem ; 274(24): 17033-41, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10358054

ABSTRACT

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A. , Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the beta2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.


Subject(s)
Evolution, Molecular , Receptors, Adrenergic, beta-2/metabolism , Receptors, Parathyroid Hormone/metabolism , Zinc/pharmacology , Animals , Binding, Competitive , GTP-Binding Protein alpha Subunits, Gs/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Histidine/genetics , Humans , Isoproterenol/metabolism , Models, Molecular , Mutation , Pindolol/analogs & derivatives , Pindolol/metabolism , Protein Engineering , Protein Structure, Secondary , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/drug effects , Receptors, Adrenergic, beta-2/genetics , Receptors, Parathyroid Hormone/chemistry , Receptors, Parathyroid Hormone/drug effects , Receptors, Parathyroid Hormone/genetics , Rod Opsins , Secretin , Sequence Alignment , Signal Transduction
12.
J Biol Chem ; 274(26): 18793-800, 1999 Jun 25.
Article in English | MEDLINE | ID: mdl-10373496

ABSTRACT

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, alphaz, which is myristoylated and palmitoylated (Morales, J., Fishburn, C. S., Wilson, P. T., and Bourne, H. R. (1998) Mol. Biol. Cell 9, 1-14). Now we extend this analysis by characterizing the mechanisms that target newly synthesized alphaz to the plasma membrane (PM) and analyze the role of lipid modifications in this process. In comparison with newly synthesized alphas, which is palmitoylated but not myristoylated, alphaz moves more rapidly to the membrane fraction following synthesis in the cytosol. Newly synthesized alphaz associates randomly with cellular membranes, but with time accumulates at the PM. Palmitoylated alphaz is present only in PM-enriched fractions, whereas a nonpalmitoylated mutant of alphaz (alphazC3A) associates less stably with the PM than does wild-type alphaz. Expression of a C-terminal fragment of the beta-adrenoreceptor kinase, which sequesters free betagamma, impairs association of both alphaz and alphazC3A with the PM, suggesting that the alpha subunit must bind betagamma in order to localize at the PM. Based on these findings, we propose a model in which, following synthesis on soluble ribosomes, myristoylated alphaz associates randomly and reversibly with membranes; upon association with the PM, alphaz binds betagamma, which promotes its palmitoylation, thus securing it in the proper place for transmitting the hormonal signal.


Subject(s)
Cell Membrane/metabolism , GTP-Binding Protein alpha Subunits , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/metabolism , Heterotrimeric GTP-Binding Proteins , Palmitates/metabolism , Animals , Biological Transport , Brefeldin A/pharmacology , CHO Cells , Cricetinae , Exocytosis , GTP-Binding Proteins/biosynthesis , Myristic Acid/metabolism , Protein Synthesis Inhibitors/pharmacology , Signal Transduction
13.
J Biol Chem ; 274(22): 15757-65, 1999 May 28.
Article in English | MEDLINE | ID: mdl-10336477

ABSTRACT

Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.


Subject(s)
Antigens, CD/chemistry , Membrane Proteins/genetics , Protein Structure, Secondary , Receptors, Complement/chemistry , Amino Acids/genetics , Antigens, CD/genetics , Complement C5a/metabolism , Evolution, Molecular , Gene Library , Humans , Membrane Proteins/chemistry , Models, Molecular , Mutation , Receptor, Anaphylatoxin C5a , Receptors, Complement/genetics , Sequence Deletion , Signal Transduction , Yeasts/genetics
14.
Mol Biol Cell ; 10(4): 1163-78, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10198064

ABSTRACT

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.


Subject(s)
Antigens, CD/physiology , Chemotaxis, Leukocyte/physiology , Neutrophils/physiology , Receptors, Complement/physiology , Receptors, Immunologic/physiology , Receptors, Peptide/physiology , Antigens, CD/genetics , Cell Adhesion , Cell Line , Cell Polarity , Chemotaxis, Leukocyte/drug effects , Complement C5a/pharmacology , Complement C5a/physiology , Green Fluorescent Proteins , Humans , Leukemia , Luminescent Proteins/genetics , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Receptor, Anaphylatoxin C5a , Receptors, Complement/genetics , Receptors, Formyl Peptide , Receptors, Immunologic/genetics , Receptors, Peptide/genetics , Recombinant Fusion Proteins/metabolism , Transfection , Tumor Cells, Cultured
16.
J Biol Chem ; 274(5): 2824-8, 1999 Jan 29.
Article in English | MEDLINE | ID: mdl-9915816

ABSTRACT

Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the Gi subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein betagamma subunits and can be triggered by agonists for receptors coupled to Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq. Because activation of any G protein presumably releases free Gbetagamma, we tested the hypothesis that chemotaxis also requires activated alpha subunits (Galphai) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Galpha, Galphaqz5, which resembles Galphai in susceptibility to activation by Gi-coupled receptors but cannot regulate the Galphai 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 Galphai but not that of Galphaqz5. We infer that chemotaxis does not require activation of Galphai. Because chemotaxis is mediated by Gbetagamma subunits released when Gi-coupled receptors activate Galphaqz5, but not when Gq- or Gs-coupled receptors activate their respective G proteins, we propose that Gi-coupled receptors transmit a necessary chemotactic signal that is independent of Galphai.


Subject(s)
Antigens, CD/metabolism , Chemotaxis, Leukocyte , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptors, Interleukin/metabolism , Adenylate Cyclase Toxin , Adenylyl Cyclases/metabolism , Carbachol/pharmacology , Cell Line , Chemotaxis, Leukocyte/drug effects , GTP-Binding Protein alpha Subunit, Gi2 , GTP-Binding Proteins/metabolism , Humans , Interleukin-8/pharmacology , Neutrophils/drug effects , Neutrophils/physiology , Pertussis Toxin , Proto-Oncogene Proteins/metabolism , Receptors, Interleukin-8A , Virulence Factors, Bordetella/pharmacology
17.
Proc Natl Acad Sci U S A ; 96(2): 499-504, 1999 Jan 19.
Article in English | MEDLINE | ID: mdl-9892662

ABSTRACT

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein alpha subunit (Galpha), thereby generating two potential signaling molecules, Galpha-GTP and free Gbetagamma. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Galpha mutation. Here we describe a mutant alpha subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the alpha subunit (alphas) of Gs three separate mutations chosen because they impair alphas function in complementary ways: the A366S mutant reduces affinity of alphas for binding GDP, whereas the G226A and E268A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active alphas mutant (alphas-R201C) or to agonists for receptors that activate Gq or Gi. This alphas triple mutant and cognate Galpha mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.


Subject(s)
GTP-Binding Proteins/genetics , Mitogen-Activated Protein Kinases , Mutation/genetics , Signal Transduction/genetics , Adenylyl Cyclase Inhibitors , Animals , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Chorionic Gonadotropin/pharmacology , Cyclic AMP/metabolism , Mitogen-Activated Protein Kinase 3 , Protein Binding/genetics , Receptors, Cell Surface/antagonists & inhibitors , Receptors, LH/genetics , Transfection/genetics
18.
J Biol Chem ; 273(44): 28597-602, 1998 Oct 30.
Article in English | MEDLINE | ID: mdl-9786851

ABSTRACT

G proteins transmit signals from cell surface receptors to intracellular effectors. The intensity of the signal is governed by the rates of GTP binding (leading to subunit dissociation) and hydrolysis. Mutants that cannot hydrolyze GTP (e.g. GsalphaQ227L, Gi2alphaQ205L) are constitutively activated and can lead to cell transformation and cancer. Here we have used a genetic screen to identify intragenic suppressors of a GTPase-deficient form of the Galpha in yeast, Gpa1(Q323L). Sequencing revealed second-site mutations in three conserved residues, K54E, R327S, and L353Delta (codon deletion). Each mutation alone results in a complete loss of the beta gamma-mediated mating response in yeast, indicating a dominant-negative mode of inhibition. Likewise, the corresponding mutations in a mammalian Gi2alpha (K46E, R209S, L235Delta) lead to inhibition of Gbeta gamma-mediated mitogen-activated protein (MAP) kinase phosphorylation in cultured cells. The most potent of these beta gamma inhibitors (R209S) has no effect on Gi2alpha-mediated regulation of adenylyl cyclase. Despite its impaired ability to release beta gamma, purified recombinant Gpa1(R327S) is fully competent to bind and hydrolyze GTP. These mutants will be useful for uncoupling Gbeta gamma- and Galpha-mediated signaling events in whole cells and animals. In addition, they serve as a model for drugs that could directly inhibit G protein activity and cell transformation.


Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , Mutation , Signal Transduction , Animals , Base Sequence , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA Primers , Escherichia coli/genetics , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Mating Factor , Mutagenesis, Site-Directed , Peptides/metabolism , Protein Conformation , Saccharomyces cerevisiae/genetics
19.
Nature ; 394(6688): 35-8, 1998 Jul 02.
Article in English | MEDLINE | ID: mdl-9665125

ABSTRACT

How does a trimeric G protein on the inside of a cell membrane respond to activation by a transmembrane receptor? G-protein mutations in patients with hypertension and inherited endocrine disorders enhance or block signals from stimulated receptors. In combination with three-dimensional crystal structures and results from biochemical experiments, the phenotypes produced by these mutations suggest a model for the molecular activation mechanism that relays hormonal and sensory signals transmitted by many transmembrane receptors.


Subject(s)
GTP-Binding Proteins/metabolism , Models, Biological , Receptors, Cell Surface/metabolism , GTP-Binding Proteins/genetics , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Models, Molecular , Mutation , Protein Conformation
20.
Mol Biol Cell ; 9(1): 1-14, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9436987

ABSTRACT

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the alpha subunits are myristoylated and/or palmitoylated, whereas the gamma chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled alpha subunit, alpha z. Wild-type alpha z (alpha z-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does alpha z-WT. Removal of the myristoylation site produced a mutant alpha z that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of beta gamma with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on alpha z-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on alpha z-WT. Together these results suggest that myristate and beta gamma promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on alpha z specific localization at the plasma membrane.


Subject(s)
GTP-Binding Protein alpha Subunits , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/physiology , Heterotrimeric GTP-Binding Proteins , Signal Transduction , Animals , Binding Sites , CHO Cells , Cell Membrane/metabolism , Cell Membrane/physiology , Cricetinae , GTP-Binding Proteins/genetics , Microscopy, Fluorescence , Myristic Acid/metabolism , Palmitates/metabolism
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