In situ binding of a photo-affinity GTP analog to synaptic membrane G-proteins. Distribution of bound GTP analog reflects the status of adenylate cyclase.
Regulation of synaptic membrane adenylate cyclase is likely to involve interaction between neurotransmitter receptors, G-proteins and the adenylate cyclase catalytic unit as well as several other membrane proteins and lipids. Despite intensive study of this system, regulation of guanine nucleotide binding by the G-proteins which stimulate [Gs] or inhibit [Gi] adenylate cyclase has been examined only when those proteins have been purified and removed from the influence of the membrane environment. The hydrolysis-resistant photoaffinity GTP-analog, P3-(4-azidoanilido)-P1 5'-GTP (AAGTP) is able to bind specifically to the G-proteins in rat cerebral cortex synaptic membranes and, in this study, we have used this probe to examine the specificity and selectivity of guanine nucleotide binding to each G-protein without removing those proteins from the synaptic membrane. Marked differences were noted between guanine nucleotide binding data obtained with detergent-soluble G-proteins and data from this in situ approach. In these studies it was found that the affinity of the G-proteins binding AAGTP correlated well with the expression of adenylate cyclase activity, the affinity of both forms of Gs increasing under conditions favoring the stimulation of that enzyme.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010777:Photochemistry; D051381:Rats; D011919:Rats, Inbred Strains; D013570:Synaptic Membranes / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases