A point mutation uncouples transducin-alpha from the photoreceptor RGS and effector proteins.
A novel gain-of-function mutation, R243Q, has been recently identified in the Candida elegans Gqalpha protein EGL-30. The position corresponding to Arg243 in EGL-30 is absolutely conserved among heterotrimeric G proteins. This mutation appears to be the first gain-of-function mutation in the switch III region of Galpha subunits. To investigate consequences of the R-->Q mutation we introduced the corresponding R238Q mutation into transducin-like Gtalpha* subunit. The mutant retained intact interactions with Gtbetagamma and rhodopsin but exhibited a twofold reduction in the kcat value for guanosine 5'-triphosphate (GTP) hydrolysis. The GTPase activity of R238Q was not accelerated by the RGS domain of the visual GTPase-activating protein, RGS9-1. In addition, R238Q displayed a significant impairment in the effector function. Our data and the crystal structures of transducin suggest that the major reason for the reduced intrinsic GTPase activity of R238Q and the lack of RGS9 function is the break of the conserved ionic contact between Arg238 and Glu39, which apparently stabilizes the transitional state for GTP hydrolysis. We hypothesize that the R243Q mutation in EGL-30 severs the ionic interaction of Arg243 with Glu43, leading to a defective inactivation of the mutant by the C. elegans RGS protein EAT-16.
Natochin
Michael
M
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA.
Artemyev
Nikolai O
NO
D000595:Amino Acid Sequence; D002175:Candida; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics; D006160:Guanosine Triphosphate / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D015293:Transducin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D020710:RGS Proteins; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015293:Transducin