| A-kinase (PKA) anchoring proteins (AKAPs) are essential for targeting cAMP and PKA (mainly PKA type II) to specific locales in the cell to control function. The purpose of this dissertation was to determine the expression, localization, and molecules associated with AKAP5, and the contribution of AKAP5 in amylase secretion from mouse parotid acini. Multiple AKAPs, i.e. AKAP5, AKAP6 and Ezrin, were identified by RII overlay analysis and immunoblot analyses. Immunocytofluorescence analyses localized AKAP5 to the basolateral membrane of the acinar cell and AKAP6 to the perinuclear region. Immunoblot analyses revealed that cAMP dependent protein kinase type II (PKA-II), PKC, and the serine/threonine phosphatase, PP2B co-immunoprecipitated with AKAP5. Additionally, AC6, but not AC3 or AC8, was found in immunoisolates of AKAP5. In functional studies, amylase release stimulated by the beta-adrenergic receptor agonist, isoproterenol, was reduced by approximately 50% in AKAP5 mutant mice. In contrast, no significant decrease in amylase secretion was noted in response to PKA activators that act downstream of the receptor, i.e. forskolin and 6-Phe-cAMP or to the Epac activator, 8-pMeOPT-2'-O-Me-cAMP. The lack of complete inhibition of isoproterenol stimulated amylase secretion was also observed in cells incubated with the AKAP inhibitor, St-Ht31 and with PKA inhibitors, PKI and H89. In cells transfected with a dominant mutant of Rac1 (Rac1 N17), a downstream effector of the cAMP/Epac/Rap1 signaling cascade, isoproterenol stimulated amylase release was reduced by 30%, whereas, 8-pMeOPT-2'-O-Me-cAMP-mediated secretion was abrogated. 8-pMeOPT-2'-O-Me-cAMP stimulated accumulation of subplasmalemmal cytoskeleton was also eliminated in cells expressing Rac1N17. In contrast, PKA mediated secretion was unaffected. Data suggest that isoproterenol stimulated amylase secretion from mouse parotid acini occurs via a PKA/AKAP5/AC6 complex, and likely involves Epac, other adenylyl cyclases and/or activation of PKA-I/PKA-II associated with other AKAPs. Rac1, which is activated by Epac, but not PKA, regulates secretion in isoproterenol stimulated cells. |
