title: Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi-mediated inhibition of cAMP production.
abstract: Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.
authors.affiliation: Institute of Pharmacology and Clinical Pharmacy, Universität Marburg, 35032 Marburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Milde
authors.forename: Andreas
authors.initials: A
authors.lastname: Rinne
authors.forename: Frank
authors.initials: F
authors.lastname: Wunder
authors.forename: Stefan
authors.initials: S
authors.lastname: Engelhardt
authors.forename: Moritz
authors.initials: M
authors.lastname: Bünemann
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis*; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; C584114:GNAI1 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; C419510:adenylyl cyclase type V