The role of Galpha(s) in heterologous sensitization of adenylyl cyclase
Abstract
Acute activation of Gαi/o-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of Gαi/o-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity following subsequent activation. Several indirect observations have suggested the involvement of enhanced Gαs adenylyl cyclase interactions in the expression of sensitization, however, evidence supporting a direct role for Gαs has not been well established. We have used several genetic approaches to further examine the role of Gαs in heterologous sensitization of adenylyl cyclase. In the first approach, Gαs insensitive mutants of AC1 (F293L and Y973S) were constructed, and these AC1 mutants retained sensitivity to Ca2+ and forskolin activation. Persistent (2 h) activation of the D2 dopamine receptor resulted in a significant augmentation of basal and drug-stimulated AC1 activity, whereas sensitization of AC1 F293L and Y973S was markedly reduced. In the second strategy, the requirement of an intact receptor-Gαs signaling pathway for the expression heterologous sensitization was examined using dominant negative Gαs mutants (α3β5 G226A/A366S or α3β5 G226A/E268A/A366S) that disrupt D1 dopamine receptor activation of AC1. D1 dopamine receptor-Gα s signaling was blocked in the presence of these Gαs mutants, but D2 agonist-induced sensitization of Ca2+-stimulated AC1 activity was not altered. Subsequent studies examined the absolute requirement of Gαs in sensitization of recombinant AC1 and AC5 using a novel Gαs-deficient (GnasE2−/E2− ) cell line. Persistent activation (18 h) of the D2 dopamine receptor markedly enhanced subsequent forskolin and Ca2+-stimulated AC1 activity in GnasE2−/E2− cells, whereas sensitization of forskolin-stimulated AC5 activity occurred only in the presence of transfected Gαs. The physiological significance of D2 dopamine receptor-induced sensitization of adenylyl cyclase was also examined in the present report. D2 dopamine receptors are colocalized with Gαs-coupled A2A adenosine receptors on GABAergic neurons in the striatum and their functional interactions have been implicated in the pharmacotherapy of Parkinson's disease. We have confirmed that acute activation of the D 2 dopamine receptor inhibits A2A adenosine receptor-stimulated cyclic AMP accumulation. In contrast, persistent activation of the D 2 dopamine receptor resulted in sensitization of neuronal A2A adenosine receptor signaling. Taken together, these observations suggest a differential role for Gαs in the expression of heterologous sensitization of adenylyl cyclase, which may be involved in neuropsychiatric disorders associated with increased D2 dopamine receptor signaling.
Degree
Ph.D.
Advisors
Watts, Purdue University.
Subject Area
Pharmacology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.