Two distinct caveolin-1 domains mediate the functional interaction of caveolin-1 with protein kinase A

Numerous components of the cAMP-based signaling cascade, namely G-proteins and G-protein coupled receptors, adenylyl cyclase, and protein kinase A (PK A) have been localized to caveolae and shown to be regulated by the caveola r marker proteins, the caveolins. In order to gain mechanistic insights int o these processes in vivo, we have assessed the functional interaction of c aveolin-1 (Cav-1) with PKA using mutational analysis. As two regions of Cav -1 had previously been implicated in PKA signaling in vitro, we constructed Cav-1 molecules with mutations/deletions in one or both of these domains. Examination of these mutants shows that Cav-1 requires the presence of eith er the scaffolding domain or the COOH-terminal domain (but not both) to fun ctionally interact with and inhibit PKA. Interestingly, in contrast to the wild-type protein, these Cav-1 mutants are not localized to caveolae microd omains. However, upon coexpression with wild-type Cav-1, a substantial amou nt of the mutants was recruited to the caveolae membrane fraction. Using th e Cav-1 double mutant with both disrupted scaffolding and COON-terminal dom ains, we show that wild-type Cav-1's inhibition of PKA signaling can be par tially abrogated in a dose-responsive manner; i.e., the mutant acts in a do minant-negative fashion. Thus, this dominant-negative caveolin-1 mutant wil l be extremely valuable for assessing the functional role of endogenous cav eolin-1 in regulating a variety of other signaling cascades.