title: Using molecular tools to dissect the role of Galphas in sensitization of AC1.
abstract: Short-term activation of Galpha(i/o)-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of Galpha(i/o)-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity after subsequent activation by Galpha(s) or forskolin. Several indirect observations have suggested the involvement of increased Galpha(s)-adenylyl cyclase interactions in the expression of sensitization; however, evidence supporting a direct role for Galpha(s) has not been well established. In the present report, we used two genetic approaches to further examine the role of Galpha(s) in heterologous sensitization of Ca(2+)-sensitive type 1 adenylyl cyclase (AC1). In the first approach, we constructed Galpha(s)-insensitive mutants of AC1 (F293L and Y973S) that retained sensitivity to Ca2+ and forskolin activation. Persistent (2 h) activation of the D2 dopamine receptor resulted in a significant augmentation of basal or Ca(2+)- and forskolin-stimulated AC1 activity; however, sensitization of Galpha(s)-insensitive mutants of AC1 was markedly reduced compared with wild-type AC1. In the second strategy, we examined the requirement of an intact receptor-Galpha(s) signaling pathway for the expression of sensitization using dominant-negative Galpha(s) mutants (alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S) to disrupt D1 dopamine receptor activation of recombinant AC1. D1 dopamine receptor-Galpha(s) signaling was attenuated in the presence of alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S, but D2 agonist-induced sensitization of Ca(2+)-stimulated AC1 activity was not altered. Together, the present findings directly support the hypothesis that the expression of sensitization of AC1 involves Galpha(s)-adenylyl cyclase interactions.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Vortherms
authors.forename: Chau H
authors.initials: CH
authors.lastname: Nguyen
authors.forename: Catherine H
authors.initials: CH
authors.lastname: Berlot
authors.forename: Val J
authors.initials: VJ
authors.lastname: Watts
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D019943:Amino Acid Substitution; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology*; D006801:Humans; D007668:Kidney; D019257:Quinpirole / Q000494:pharmacology; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; C113776:dopamine D2L receptor; D019257:Quinpirole; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases