A subset of growth hormone-secreting human pituitary tumours carries somatic mutations that inhibit GTPase activity of a G protein alpha chain, alpha(s). The resulting activation of adenylyl cyclase bypasses the cells' normal requirement for trophic hormone. Amino acids substituted in the putative gsp oncogene identify a domain of G protein alpha-chains required for intrinsic ability to hydrolyse GTP. This domain may serve as a built-in counter-part of the separate GTPase-activating proteins required for GTP hydrolysis by small GTP-binding proteins such as p21ras.
Membrane-bound G proteins carry information from receptors on the outside of cells to effector proteins inside cells. The alpha subunits of these heterotrimeric proteins bind and hydrolyse GTP and control the specificity of interactions with receptor and effector elements. Signalling by G proteins involves a cycle in which the inactive alpha beta gamma-GDP complex dissociates to produce alpha*-GTP, which is capable of activating the effector enzyme or ion channel; the alpha*-GTP complex hydrolyses bound GTP and reassociates with beta gamma to form the inactive complex. We have characterized a mutation that interrupts this GTP-driven cycle in alpha s, the alpha-chain of Gs, the G protein that stimulates adenylyl cyclase. The mutation converts a glycine to an alanine residue in the presumed GDP-binding domain of alpha s. The location and biochemical consequences of this mutation suggest a common mechanism by which binding of GTP or ATP may induce changes in the conformation of a number of nucleoside triphosphate binding proteins.
The mammalian G proteins transduce information from extracellular signals, including neurotransmitters, hormones and sensory stimuli, into regulation of effector enzymes or ion channels within cells. Triggered by appropriate extracellular signals, receptor proteins specifically activate members of the G protein family by catalysing replacement of GDP by GTP at the guanine nucleotide binding site. Like the receptor proteins, the heterotrimeric G proteins exhibit impressive structural similarities, suggesting that all receptor-G protein interactions use homologous structural elements and a single molecular mechanism. Topologically equivalent portions of each G protein may therefore interact with the appropriate receptor. We recently predicted the secondary structure of a composite G protein alpha-chain and proposed that a predicted amphipathic alpha-helix at the extreme carboxy-terminus of the polypeptide directly contacts receptors. This proposal has now been confirmed by sequencing complementary DNAs of the gene that encodes the alpha-chain (alpha s) of the stimulatory regulator (Gs) of adenylyl cyclase in wild-type cells and in a mutant mouse S49 lymphoma cell line, unc, in which Gs cannot be activated by hormone receptors. The sequences reveal a point mutation in the unc gene that substitutes a proline residue for an arginine near the carboxy-terminus of the alpha s-polypeptide. Expression of recombinant alpha s-unc in genetically alpha s-deficient S49 cells reproduces the unc phenotype.
The G protein family of signal transducers includes five heterotrimers, which are most clearly distinguished by their different a chains. The family includes G, and G1, the stimulatory and inhibitory GTP-binding regulators of adenylate cyclase; G., a protein of unknown function abundant in brain; and transducin 1 and transducin 2, proteins involved in retinal phototransduction. Using a bovine at, cDNA as a hybridization probe, we have isolated mouse cDNAs that encode a chains of two G proteins. One encodes a polypeptide of 377 amino acids (Mr 43,856), identified as a. because it specifically fails to hybridize with any transcript in an a,-deficient S49 mouse lymphoma mutant, cyc-; the other encodes a polypeptide of 355 amino acids (Mr 40,482), presumed to be aj. These a chains and those of the retinal transducins exhibit impressive sequence homology; Of the four, ati and at2 are most alike (81% identical amino acid residues), whereas the presumptive aj is more similar than a. to at, (63% vs. 38% identical residues). Sequence homologies with p2lrM and elongation factor Tu identify regions of the a chains that form the site for GTP binding and hydrolysis. Further comparison of the a-chain sequences suggests additional regions that may contribute to interactions with ,By subunits and the receptor and effector components of different signal transduction systems. 6) and ion channels (7-9). All of the G proteins are heterotrimers, with virtually identical A chains, similar y chains, and distinctive a chains. The a chains, with Mrs ranging from 39,000 to 45,000, bind and hydrolyze GTP and serve as substrates for ADP-ribosylation by the exotoxins of Vibrio cholerae (in G, and transducin 1) and Bordetella pertussis (in Gi, Go, and transducin 1). In the GTP-bound form, the a chains of G, (a,) and transducin 1 (at,) activate their respective effector enzymes, adenylate cyclase and cGMP phosphodiesterase (for review, see refs. 1, 4).Using a cDNA encoding bovine at, (10) as a hybridization probe, we have isolated and sequenced murine cDNAs that encode a, and a second a chain, presumed to be aj. Homologies and differences among the deduced amino acid sequences of the G protein and transducin a chains point to specific regions that may interact with guanine nucleotides, receptors, effector enzymes, and the G protein /3y complex.MATERIALS AND METHODS Detection of cDNA Inserts. David Goeddel (Genentech) provided a XgtlO cDNA library prepared by using RNA from the murine macrophage cell line PU-5 (11). We screened this library for plaques that hybridized (12) to the bovine at, cDNA (10) at low stringency [in 35% formamide, 0.75 M NaCl, 75 mM sodium citrate, S x concentrated Denhardt's solution (0.02% bovine serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone), 0.5% NaDodSO4, 0.5 mg of denatured salmon sperm DNA per ml, and 5 x 105 cpm of probe per ml for 48 hr at 42°C] (13). Filters were washed three times for 5 min at 25°C in 0.3 M NaCl/30 mM sodium citrate/0.1% NaDodSO4 and twice for 30 min at 50°C in 0.15 M NaCl/...
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