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Biomedical subjects

H R Bourne

Publications and source records attributed to H R Bourne.

At least 19 recordsLinked to original sources
PubMed1997

Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin.

Transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have revealed contact surfaces between G protein subunits, but not the surfaces or molecular mechanism through which Galphabetagamma responds to activation by transmembrane receptors. Such a surface was identified from the results of testing 100 mutant alpha subunits of the retinal G protein transducin for their ability to interact with rhodopsin. Sites at which alanine substitutions impaired this interaction mapped to two distinct Galpha surfaces: a betagamma-binding surface and a putative receptor-interacting surface. On the basis of these results a mechanism for receptor-catalyzed exchange of guanosine diphosphate for guanosine triphosphate is proposed.

Aluminum Compounds↗
PubMed1996

Reciprocal regulation of Gs alpha by palmitate and the beta gamma subunit.

Hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase, promotes dissociation of alpha s from G beta gamma, accelerates removal of covalently attached palmitate from the G alpha subunit, and triggers release of a fraction of alpha s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant alpha s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated alpha s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound beta gamma with 5-fold higher affinity. beta gamma protected GDP-bound alpha s, but not alpha s-GTP[gamma S], from depalmitoylation by a recombinant esterase. We conclude that beta gamma binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of alpha s and that dissociation of alpha s.GTP from beta gamma is likely to mediate receptor-induced alpha s depalmitoylation and translocation of the protein to cytosol in intact cells.

Animals↗
PubMed1996

Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F.

A large superfamily of receptors containing seven transmembrane (TM) helices transmits hormonal and sensory signals across the plasma membrane to heterotrimeric G proteins at the cytoplasmic face of the membrane. To investigate how G-protein-coupled receptors work at the molecular level, we have engineered metal-ion-binding sites between TM helices to restrain activation-induced conformational change in specific locations. In rhodopsin, the photoreceptor of retinal rod cells, we substituted histidine residues for natural amino acids at the cytoplasmic ends of the TM helices C and F. The resulting mutant proteins were able to activate the visual G protein transducin in the absence but not in the presence of metal ions. These results indicate that the TM helices C and F are in close proximity and suggest that movements of these helices relative to one another are required for transducin activation. Thus a change in the orientations of TM helices C and F is likely to be a key element in the mechanism for coupling binding of ligands (or isomerization of retinal) to the activation of G-protein-coupled receptors.

Amino Acid Sequence↗
PubMed1996

Galpha12 and Galpha13 regulate extracellular signal-regulated kinase and c-Jun kinase pathways by different mechanisms in COS-7 cells.

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.

Animals↗
PubMed1996

Pseudohypoparathyroidism, a novel mutation in the betagamma-contact region of Gsalpha impairs receptor stimulation.

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.

Adenylyl Cyclases↗
PubMed1996

Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.

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.

Amino Acid Sequence↗
PubMed1996

An evolutionary trace method defines binding surfaces common to protein families.

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 experimental 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 delineates the functional epitope and identifies residues critical to 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 recognition. More generally, it provides an evolutionary perspective for judging the functional or structural role of each residue in protein structure.

Amino Acid Sequence↗
PubMed1996

Activation-induced subcellular redistribution of Gs alpha.

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.

Adrenergic beta-Agonists↗
PubMed1996

Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation.

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.

Animals↗
PubMed1995

Solution structure of the GTPase activating domain of alpha s.

We have used heteronuclear three-dimensional NMR spectroscopy to determine the solution structure of a 141 residue protein containing the GTPase activating domain from the alpha chain of the heterotrimeric G protein Gs. The domain contains six alpha-helices and is stable and structured in solution despite having been excised from the intact Gs protein. The N-terminal ten and C-terminal 11 residues of the protein are unstructured in solution while the core is well determined by the 2483 distance and torsion restraints derived from the NMR spectra. The final ensemble of 14 structures, generated with a hybrid distance geometry/simulated annealing protocol, have an average to-the-mean backbone root-mean-square deviation of 0.39 A for the core residues 89 to 201. The majority of the structure is remarkably similar to that observed for the cognate domains in crystal structures of the homologous proteins alpha t and alpha i1. However, the orientations of the second helix and the subsequent interhelical loops differ markedly among the three proteins. This structural divergence, along with functional studies of chimeric proteins, suggests that this region of the domain interacts with either the downstream effector adenylyl cyclase or with some other intermediary protein.

Amino Acid Sequence↗
PubMed1995

GTPases: a family of molecular switches and clocks.

Members of the GTPase superfamily share a core domain with a conserved three-dimensional structure and a common GTPase cycle, but perform a wide variety of regulatory tasks in eukaryotic cells. Evolution has created functional diversity from the conserved GTPase structure in two principal ways: (i) by combining in the product of a single gene the core GTPase domain attached to one or more additional folded domains; (ii) by building around a core GTPase an assembly of proteins encoded by different genes. Analysis of the patterns of conserved amino acid side chains on surfaces of G alpha proteins reveals interfaces with other proteins in the G-protein signal linking device.

Animals↗
PubMed1995

Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells.

We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha 1) of retinal transducin (Gt, also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of Gt (alpha t, beta 1 and gamma 1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of Gt, as measured by the ability of GTP gamma S to protect alpha t fragments from proteolytic degradation. The activation of Gt is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating Gt show similar defects in this assay. alpha t mutations that mimic the corresponding mutations in the alpha-subunit of Gs also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha t proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha 1 to activation by rhodopsin. All four mutants retain their ability to be activated by AIF-4. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha t with rhodopsin.

Amino Acid Sequence↗
PubMed1995

Mutant alpha subunit of Gz transforms Swiss 3T3 cells.

Many hormones and neurotransmitters induce cell proliferation by regulating signaling pathways controlled by heterotrimeric G proteins. Mutations that activate the alpha subunits of Gs and Gi2 produce the gsp and gip2 oncogenes that are found in certain human endocrine tumors. Similar mutations have conferred on other G alpha subunits the ability to promote neoplastic transformation in cultured mammalian cells. Gz, a G protein whose normal signaling function is poorly understood, shares with Gi2 the ability to inhibit adenylyl cyclase. We asked whether mutationally activated alpha z can stimulate cell proliferation in a cell line in which stimulation adenylyl cyclase is mitogenic, Swiss 3T3 cells. Stable expression of alpha z-Q205L in Swiss 3T3 cells induced focus formation, a faster growth rate with a higher saturation density, anchorage-independent growth in soft agar, and increased [3H]thymidine incorporation in the absence of growth factors. alpha z-Q205L produced a similar but less extensively transformed phenotype in NIH3T3 cells--increased saturation density in culture, a smaller number of foci and few colonies in soft agar. Stimulation of thymidine incorporation by alpha z-Q205L in Swiss 3T3 cells was increased by co-treatment with cholera toxin, a stimulator of adenylyl cyclase. Taken together, our results indicate that alpha z stimulates one or more mitogenic pathways in Swiss 3T3 cells, and that effectiveness of these mitogenic pathways does not require reducing the concentration of cellular cAMP.

3T3 Cells↗
PubMed1995

Fatty acylation of alpha z. Effects of palmitoylation and myristoylation on alpha z signaling.

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.

Acylation↗
PubMed1995

Potentiation of Gi-mediated phospholipase C activation by retinoic acid in HL-60 cells. Possible role of G gamma 2.

Differentiated HL-60 cells acquire responsiveness to fMet-Leu-Phe (fMLP), which activates phospholipase C and O2- generation in a pertussis toxin-sensitive manner. Addition of retinoic acid (RA) for the last 24 h during dimethyl sulfoxide (Me2SO)-induced differentiation enhanced fMLP-dependent signals and interaction between fMLP receptor and G(i). RA modifies both the function and subunit composition of G(i)2, the predominant G(i) of HL-60 membranes, as shown by comparing purified G(i)2 from membranes of Me2SO-treated cells (D-G(i)2) to G(i)2 from membranes of cells treated with both Me2SO and RA (DR-G(i)2). As compared to D-G(i)2, DR-G(i)2 induced more fMLP binding when added to membranes of pertussis toxin-treated HL-60 cells and, in the presence of GTP gamma S, stimulated beta gamma-sensitive phospholipase C in extracts of HL-60 cells to a much greater extent at a lower concentrations. Immunoblasts revealed that RA induced expression of the gamma 2 subunit, which was otherwise undetectable in G(i)2 purified from HL-60 cells or in HL-60 membranes. Possibly by inducing expression of gamma 2, RA alters two functions of the G(i) beta gamma subunit, modulation of fMLP receptor-G(i)2 coupling and activation of the effector, Phospholipase C.

Cell Differentiation↗
PubMed1995

Lipid modifications of trimeric G proteins.

G protein alpha subunits and beta gamma dimers are covalently modified by lipids. The emerging picture is one in which attached lipids provide more than just a nonspecific "glue" for sticking G proteins to membranes. We are only beginning to understand how different lipid modifications of different G protein subunits affect specific protein-protein interactions and localization to specific cellular sites. In addition, regulation of these modifications, particularly palmitoylation, can provide new ways to regulate signals transmitted by G proteins.

Amino Acid Sequence↗