Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4.
Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.
Bahia
Daljit S
DS
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Sartania
Nana
N
Ward
Richard J
RJ
Cavalli
Antonella
A
Jones
Teresa L Z
TL
Druey
Kirk M
KM
Milligan
Graeme
G
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010169:Palmitic Acids / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D020710:RGS Proteins / Q000235:genetics*; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013552:Swine; D014162:Transfection
C547768:ADRA2A protein, human; D000316:Adrenergic alpha-Agonists; D020690:GTPase-Activating Proteins; D010169:Palmitic Acids; D020710:RGS Proteins; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; C098799:RGS4 protein; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D003545:Cysteine