Using Molecular Tools to Dissect the Role of G s in Sensitization of AC1

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 Ca -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 Ca and forskolin activation. Persistent (2 h) activation of the D2 dopamine receptor resulted in a significant augmentation of basal or Ca 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 receptorG s signaling pathway for the expression of sensitization using dominant-negative G s mutants ( 3 5 G226A/A366S or 3 5 G226A/E268A/A366S) to disrupt D1 dopamine receptor activation of recombinant AC1. D1 dopamine receptor-G s signaling was attenuated in the presence of 3 5 G226A/A366S or 3 5 G226A/E268A/A366S, but D2 agonist-induced sensitization of Ca -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. 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 increase in adenylyl cyclase activity after subsequent activation by G s or forskolin. This heterologous sensitization of adenylyl cyclase was described originally as a cellular model for opioid dependence and withdrawal (Sharma et al., 1975) and represents a common adaptive response to persistent activation of multiple G i/o-coupled receptors, including the D2 dopamine receptor (Watts, 2002). The molecular mechanisms involved in heterologous sensitization of adenylyl cyclase can be described by the early events (development) associated with activation of G i/o-coupled receptors and the late events (expression) associated with the activation of adenylyl cyclase by G s, forskolin, or isoform-selective activators such as Ca for type 1 adenylyl cyclase (AC1) (Watts, 2002). The development of heterologous sensitization of adenylyl cyclase is prevented by pertussis toxin treatment, implicating a role for G i/o proteins (Watts and Neve, 1996; Rhee et al., 2000). In addition, sequestration of free G subunits, liberated from activated G i/o-coupled receptors, can also block the development of heterologous sensitization of select adenylyl cyclase isoforms (i.e., AC5 and AC6) (Thomas and Hoffman, 1996; Rhee et al.,