Cytoskeletal components regulate expression of caveolae, caveolin phosphorylation and cAMP production

The cytoskeleton regulates plasma membrane topography but its role in compartmentation of caveolar‐resident signaling components involved in cAMP production has not been defined. We hypothesized that microtubules and microfilaments influence localization of caveolin (Cav) and expression of caveolae, thereby altering the distribution of G protein‐coupled receptor (GPCR) signaling components involved in cAMP production. Inhibition of microtubules with colchicine (Colch, for 60 min) or of microfilaments with cytochalasin D (CD, for 90 min) dramatically reduced the presence of Cav‐3 in buoyant membrane fractions (BF) and increased the expression of Cav‐3 in “heavy” fractions (HF) following sucrose density fractionation. Colch or CD treatment redistributed Cav‐1, Cav‐2, β 1 ‐adrenergic receptor [AR], β 2 ‐AR, Gα s and AC 5/6 from BF to HF and increased isoproterenol (β‐AR)‐stimulated cAMP production. Immunofluorescence microscopy showed that Colch decreased co‐localization of Cav‐3 and α‐tubulin and that Colch and CD decreased co‐localization between Cav‐3 and filamin. Colch and CD decreased phosphorylation of Cav‐1, Src, and p38 MAP kinase and reduced the number of caveolae/μm sarcolemma, as measured by electron microscopy. These results implicate microtubules and microfilaments in the expression of caveolae and for phosphorylation of caveolin in the blunting of GPCR‐AC signal transduction.