Rhodopsin recognition by mutant G(s)alpha containing C-terminal residues of transducin.
The C-terminal regions of the heterotrimeric G protein alpha-subunits play key roles in selective activation of G proteins by their cognate receptors. In this study, mutant G(s)alpha proteins with substitutions by C-terminal residues of transducin (G(t)alpha) were analyzed for their interaction with light-activated rhodopsin (R*) to delineate the critical determinants of the G(t)alpha/R* coupling. In contrast to G(s)alpha, a chimeric G(s)alpha/G(t)alpha protein containing only 11 C-terminal residues from transducin was capable of binding to and being potently activated by R*. Our results suggest that Cys(347) and Gly(348) are absolutely essential, whereas Asp(346) is more modestly involved in the G(t) activation by R*. In addition, the analysis of the intrinsic nucleotide exchange in mutant G(s)alpha indicated an interaction between the C terminus and the switch II region in G(t)alpha.GDP. Mutant G(s)alpha containing the G(t)alpha C terminus and substitutions of Asn(239) and Asp(240) (switch II) by the corresponding G(t)alpha residues, Glu(212) and Gly(213), displayed significant reductions in spontaneous guanosine 5'-O-(3-thiotriphosphate)-binding rates to the levels approaching those in G(t)alpha. Communication between the C terminus and switch II of G(t)alpha does not appear essential for the activational coupling between G(t) and R*, but may represent one of the mechanisms by which Galpha subunits control intrinsic nucleotide exchange.
Natochin
M
M
Department of Physiology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Muradov
K G
KG
McEntaffer
R L
RL
Artemyev
N O
NO
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D019205:GTP-Binding Protein alpha Subunits, Gs; D015293:Transducin; D005998:Glycine