Abstract:
The main membrane proteins of caveolae (caveolin-1, -2 and -3) oligomerize within lipid rich
domains to form regular invaginations of smooth muscle plasma membrane and participate in
receptor internalization and desensitization independent of clathrin-coated vesicle endocytosis. We
have previously shown that Gs-coupled VIP/PACAP receptors, VPAC2, predominantly expressed
in smooth muscle cells of the gut, are exclusively phosphorylated by GRK2 leading to receptor
internalization and desensitization. Herein, we characterized the role of caveolin-1 in VPAC2
receptor internalization and desensitization in gastric smooth muscle using three approaches: (i)
methyl ?-cyclodextrin (M?CD) to deplete cholesterol and disrupt caveolae in dispersed muscle
cells, (ii) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and (iii)
caveolin-1 knockout mice (caveolin-1?/?). Pretreatment of gastric muscle cells with VIP
stimulated tyrosine phosphorylation of caveolin-1, and induced VPAC2 receptor internalization
(measured as decrease in 125I-VIP binding after pretreatment) and desensitization (measured as
decrease in VIP-induced cAMP formation after pretreatment). Caveolin-1 phosphorylation, and
VPAC2 receptor internalization and desensitization were blocked by disruption of caveolae with
M?CD, suppression of caveolin-1 with caveolin-1 siRNA or inhibition of Src kinase activity by
PP2. Pretreatment with VIP significantly inhibited adenylyl cyclase activity and muscle relaxation
in response to subsequent addition of VIP in freshly dispersed muscle cells and in muscle strips
isolated from wild type and caveolin-1?/?mice; however, the inhibition was significantly
attenuated in caveolin-1?/? mice. These results suggest that caveolin-1 plays an important role in
VPAC2 receptor internalization and desensitization.