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Biomedical subjects

A G Gilman

Publications and source records attributed to A G Gilman.

At least 19 recordsLinked to original sources
PubMed2001

Cross talk: interview with Al Gilman.

Alfred Goodman Gilman was born in the same year (1941) that his father and Louis Goodman published the first edition of The Pharmacological Basis of Therapeutics. Pharmacology has thus always been part of his life, and in his own career he has focused primarily on cell signaling. For the past twenty years, he has chaired the Department of Pharmacology at UT Southwestern, and his long list of accomplishments includes a Nobel Prize (1994) for his work on G proteins. In 1998, Gilman embarked on his most ambitious program of research yet, bringing dozens of leading investigators from the cell signaling community to Dallas in order to plan out a ten-year project aiming "to understand as completely as possible the relationships between sets of inputs and outputs in signaling cells." Now directing the full-fledged, federally funded Alliance for Cellular Signaling, Gilman stresses that a solid database for constructing a "virtual cell" will depend on extensive collaboration from the entire signaling community. (For a complete Program Summary, and to register for membership in the Alliance, consult www.cellularsignaling.org.) The luminaries that were invited to the Dallas planning meeting, in fact, were greeted at the door with a note from Gilman exhorting them: please check EGO at door.

History, 20th Century↗
PubMed2000

Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase.

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G(s)alpha. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C(1) and C(2)) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the beta4-beta5 loop of the C(2) domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C(1) domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C(2) domain of adenylyl cyclase for the wild type C(1) domain was exploited to isolate a complex containing VC(1), IIC(2), and G(s)alpha-guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in the absence of forskolin and a complex of VC(1), IIC(2), forskolin, and P-site inhibitor in the absence of G(s)alpha-GTPgammaS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G(s)alpha.

Adenylyl Cyclases↗
PubMed2000

Molecular basis for P-site inhibition of adenylyl cyclase.

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.

Adenylyl Cyclase Inhibitors↗
PubMed1999

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2.

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.

Aluminum Compounds↗
PubMed1999

Regulators of G protein signaling 6 and 7. Purification of complexes with gbeta5 and assessment of their effects on g protein-mediated signaling pathways.

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein beta subunits, and certain biochemical properties of RGS6/beta5 and RGS7/beta5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gbeta5 subunit (but not Gbetas 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/beta5 in that they act as GTPase-activating proteins specifically toward Galpha(o). Unlike conventional G(betagamma) complexes, RGS6/beta5 and RGS7/beta5 do not form heterotrimeric complexes with either Galpha(o)-GDP or Galpha(q)-GDP. Neither RGS6/beta5 nor RGS7/beta5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-beta1 or -beta2. However, the RGS/beta5 complexes inhibited beta(1)gamma(2)-mediated activation of phospholipase C-beta2. RGS/beta5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G(i) and G(o) by binding to phospholipase C and stimulating the GTPase activity of Galpha(o).

Amino Acid Sequence↗
PubMed1999

A candidate target for G protein action in brain.

An effector candidate for G protein action, GRIN1, was identified by screening a cDNA expression library with phosphorylated GTPgammaS-G(z)alpha as a probe. GRIN1 is a novel protein without substantial homology to known protein domains. It is expressed largely in brain and binds specifically to activated G(z)alpha, G(o)alpha, and G(i)alpha through its carboxyl-terminal region. The protein KIAA0514 (GRIN2) is homologous to GRIN1 at its carboxyl terminus and also binds to activated G(o)alpha. Both GRIN1 and G(o)alpha are membrane-bound proteins that are enriched in the growth cones of neurites. Coexpression of GRIN1 or GRIN2 with activated G(o)alpha causes formation of a network of fine processes in Neuro2a cells, suggesting that these pathways may function downstream of G(o)alpha to control growth of neurites.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Two-metal-Ion catalysis in adenylyl cyclase.

Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor.

Adenosine Triphosphate↗
PubMed1999

Modulation of the affinity and selectivity of RGS protein interaction with G alpha subunits by a conserved asparagine/serine residue.

The crystal structure of the complex between a G protein alpha subunit (Gi alpha 1) and its GTPase-activating protein (RGS4) demonstrated that RGS4 acts predominantly by stabilization of the transition state for GTP hydrolysis [Tesmer, J. J., et al. (1997) Cell 89, 251]. However, attention was called to a conserved Asn residue (Asn128) that could play a catalytic role by interacting, directly or indirectly, with the hydrolytic water molecule. We have analyzed the effects of several disparate substitutions for Asn128 on the GAP activity of RGS4 toward four G alpha substrates (Go, Gi, Gq, and Gz) using two assay formats. The results substantiate the importance of this residue but indicate that it is largely involved in substrate binding and that its function may vary with different G alpha targets. Various mutations decreased the apparent affinity of RGS4 for substrate G alpha proteins by several orders of magnitude, but had variable and modest effects on maximal rates of GTP hydrolysis when tested with different G alpha subunits. One mutation, N128F, that differentially decreased the GAP activity toward G alpha i compared with that toward G alpha q could be partially suppressed by mutation of the nearby residue in G alpha i to that found in G alpha q (K180P). Detection of GAP activities of the mutants was enhanced in sensitivity up to 100-fold by assay at steady state in proteoliposomes that contain heterotrimeric G protein and receptor.

Animals↗
PubMed1999

Persistent membrane association of activated and depalmitoylated G protein alpha subunits.

Heterotrimeric signal-transducing G proteins are organized at the inner surface of the plasma membrane, where they are positioned to interact with membrane-spanning receptors and appropriate effectors. G proteins are activated when they bind GTP and inactivated when they hydrolyze the nucleotide to GDP. However, the topological fate of activated G protein alpha subunits is disputed. One model declares that depalmitoylation of alpha, which accompanies activation by a receptor, promotes release of the protein into the cytoplasm. Our data suggest that activation of G protein alpha subunits causes them to concentrate in subdomains of the plasma membrane but not to be released from the membrane. Furthermore, alpha subunits remained bound to the membrane when they were activated with guanosine 5'-(3-O-thio)triphosphate and depalmitoylated with an acyl protein thioesterase. Limitation of alpha subunits to the plasma membrane obviously restricts their mobility and may contribute to the efficiency and specificity of signaling.

Animals↗
PubMed1999

The interactions of adenylate cyclases with P-site inhibitors.

Recent kinetic, binding and crystallographic studies using P-site inhibitors of mammalian adenylate bases provide new insights into the catalytic mechanism of these highly regulated enzymes. Here, Carmen Dessauer and colleagues discuss the conformational states of adenylate cyclase, the structural determinants of inhibitor binding and the potential uses of these inhibitors as pharmacological agents.

Adenylyl Cyclase Inhibitors↗
PubMed1998

A G protein gamma subunit-like domain shared between RGS11 and other RGS proteins specifies binding to Gbeta5 subunits.

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the alpha subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein gamma subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different Gbeta subunits reveals specific interaction between RGS11 and Gbeta5. The expression of mRNA for RGS11 and Gbeta5 in human tissues overlaps. The Gbeta5/RGS11 heterodimer acts as a GAP on Galphao, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for Galpha proteins and form complexes with specific Gbeta subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.

Amino Acid Sequence↗
PubMed1998

Sites important for PLCbeta2 activation by the G protein betagamma subunit map to the sides of the beta propeller structure.

The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.

Adenylyl Cyclases↗
PubMed1998

Identification of a Gialpha binding site on type V adenylyl cyclase.

The stimulatory G protein alpha subunit Gsalpha binds within a cleft in adenylyl cyclase formed by the alpha1-alpha2 and alpha3-beta4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory alpha subunit Gialpha could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5'-3-O-(thio)triphosphate-Gialpha1 forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by Gialpha1. These mutations suggest binding of Gialpha within the cleft formed by the alpha2 and alpha3 helices of C1, analogous to the Gsalpha binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by Gialpha. The C1b domain of the type V enzyme contributed to affinity for Gialpha, but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein alpha subunits.

Adenylyl Cyclases↗
PubMed1998

Dynamic regulation of RGS2 suggests a novel mechanism in G-protein signaling and neuronal plasticity.

Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein, and recent studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction.

Animals↗
PubMed1998

The A326S mutant of Gialpha1 as an approximation of the receptor-bound state.

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their alpha subunits. In search of an approximation of the receptor-alpha subunit complex, we have considered the properties of A326S Gialpha1, a mutation discovered originally in Gsalpha (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the alphai1beta1gamma2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Gialpha1 for GTPgammaS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the alpha subunit. The mutation also uncouples two activities of betagamma: stabilization of the GDP-bound alpha subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Gialpha1, beta gamma prevents irreversible inactivation of the alpha subunit at 30 degreesC. However, the mutation accelerates irreversible inactivation of alpha at 37 degreesC despite the presence of beta gamma. Structurally, the mutation weakens affinity for GTPgammaS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Gialpha1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Gialpha1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Galpha subunit in the GDP-bound state.

Animals↗
PubMed1998

GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain.

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Sites for Galpha binding on the G protein beta subunit overlap with sites for regulation of phospholipase Cbeta and adenylyl cyclase.

Heterotrimeric G proteins, composed of alpha and betagamma subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free betagamma activates downstream targets, but its action is terminated by association with GDP-liganded alpha subunits. Because alpha can inhibit activation of many effectors by betagamma, it is likely that the alpha subunit binding surfaces on betagamma overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on beta shown to contact alpha in the recently published crystal structures of the alphabetagamma heterotrimer (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The alpha subunit binds to the flat, top surface of the toroidal beta subunit and also extends a helix along the side of the beta subunit at blade 1. We mutated four residues on the top surface of beta (Hbeta1[L117A], Hbeta1[D228R], Hbeta1[D246S], and Hbeta1[W332A]) and two residues on the side of beta that contacts alpha (Hbeta1[N88A/K89A]). Each of the mutant proteins was able to form beta gamma dimers, but they differed in their ability to bind alpha and to activate phospholipase C beta2 (PLCbeta2), PLCbeta3, and adenylyl cyclase II. Mutation of residues along the side of the torus at blade 1 diminish affinity for alpha but do not prevent activation of any of the effectors. Mutations on the alpha binding surface differentially affected PLCbeta2, PLCbeta3, and adenylyl cyclase II. Residues that affect PLCbeta and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the alpha subunit, make many contacts on the top surface. None of the mutations affected the ability of betagamma to inhibit adenylyl cyclase I. We conclude that alpha, PLCbeta2, PLCbeta3, and adenylyl cyclase II share an interaction on the top surface of beta. The importance of individual residues is different for alpha binding and for effector activation and differs even between closely related isoforms of the same effector.

Adenylyl Cyclases↗