52 research outputs found
Gz, a guanine nucleotide-binding protein with unique biochemical properties
Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26- kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP- ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min^-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min^-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family
G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high- affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP- ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases
The Molecular Pharmacology of G Protein Signaling Then and Now: A Tribute to
ABSTRACT The recent, unfortunate death of Alfred G. ("Al") Gilman, M.D., Ph.D., represents a sad signpost for an era spanning over 40 years in molecular pharmacology. Gilman's discoveries, influence, and persona were dominant forces in research and training in pharmacology. Here, we review the progression of ideas and knowledge that spawned early work by Gilman and collaborators (among them, one of the authors) and later efforts (including those of the other author) that have recently yielded a comprehensive and precise structural understanding of fundamental topics in pharmacology: the binding of ligands to G proteincoupled receptors (GPCRs) and the interaction of GPCRs with heterotrimeric G proteins and effector molecules. Those data provide new and important insights into the molecular basis that underlies affinity and efficacy, two of the most important features of drug action, which represent the latest chapter in the saga that Al Gilman's work helped launch
The structure of the G protein heterotrimer Giα1β1γ2
AbstractThe crystallographic structure of the G protein heterotrimer Giα1(GDP)β1γ2 (at 2.3 A) reveals two nonoverlapping regions of contact between α and β, an extended interface between β and nearly all of γ, and limited interaction of α with γ. The major α/β interface covers switch II of α, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with α-GDP) explain stabilization of the inactive conformation of α by βγ. Repeated WD motifs in β form a circularized sevenfold β propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade
Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits
Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC- gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC- beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC- beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway
Farmacologia no século XX: a ciência dos medicamentos a partir da análise do livro de Goodman e Gilman
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