title: A dominant-negative Galpha mutant that traps a stable rhodopsin-Galpha-GTP-betagamma complex.
abstract: Residues comprising the guanine nucleotide-binding sites of the α subunits of heterotrimeric (large) G-proteins (Gα subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both Gα subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric Gα subunit (designated αT*) that is mainly comprised of the α subunit of the retinal G-protein transducin and a limited region from the α subunit of Gi1. The αT*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type αT*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on αT*(S43N). The αT*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type αT*. Thus, GTP-bound αT*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. αT*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type αT*. Activation of the PDE by αT*(S43N) was unaffected if either R* or β1γ1 alone was present, whereas it was inhibited when R* and the β1γ1 subunit were added together. Overall, our studies suggest that the S43N substitution on αT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound Gα subunit, and the β1γ1 complex.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-6401, USA.
authors.forename: Sekar
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cerione
mesh_terms: D000818:Animals; D002417:Cattle; D005454:Fluorescence Polarization; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D066298:In Vitro Techniques; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism*; D015293:Transducin / Q000235:genetics / Q000378:metabolism*
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D043562:Receptors, G-Protein-Coupled; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D015293:Transducin