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Using Molecular Tools to Dissect the Role of G_s in Sensitization of AC1

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (T.A.V, C.H.N., V.J.W.); and Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania (C.H.B.)

Short-term activation of G_i/o-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of G_i/o-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity after subsequent activation by G_s or forskolin. Several indirect observations have suggested the involvement of increased G_s-adenylyl cyclase interactions in the expression of sensitization; however, evidence supporting a direct role for G_s has not been well established. In the present report, we used two genetic approaches to further examine the role of G_s in heterologous sensitization of Ca²⁺-sensitive type 1 adenylyl cyclase (AC1). In the first approach, we constructed G_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 Ca²⁺- and forskolin-stimulated AC1 activity; however, sensitization of G_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-G_s signaling pathway for the expression of sensitization using dominant-negative G_s mutants (35 G226A/A366S or 35 G226A/E268A/A366S) to disrupt D₁ dopamine receptor activation of recombinant AC1. D₁ dopamine receptor-G_s signaling was attenuated in the presence of 35 G226A/A366S or 35 G226A/E268A/A366S, but D₂ agonist-induced sensitization of Ca²⁺-stimulated AC1 activity was not altered. Together, the present findings directly support the hypothesis that the expression of sensitization of AC1 involves G_s-adenylyl cyclase interactions.

Address correspondence to: Dr. Val J. Watts, Purdue University, Medicinal Chemistry and Molecular Pharmacology, 575 Stadium Mall Dr., RHPH 224A West Lafayette, IN 47907. E-mail: wattsv{at}pharmacy.purdue.edu