Journal
PEPTIDES
Volume 43, Issue -, Pages 137-145Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.peptides.2013.03.008
Keywords
Caveolin-1; Endocytosis; G protein coupled receptor; GRK2
Funding
- National Institute of Diabetes and Digestive and Kidney Diseases [DK28300]
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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 G(s)-coupled VIP/PACAP receptors, VPAC(2), 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 VPAC(2) receptor internalization and desensitization in gastric smooth muscle using three approaches: (i) methyl beta-cyclodextrin (M beta 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 VPAC(2) receptor internalization (measured as decrease in I-125-VIP binding after pretreatment) and desensitization (measured as decrease in VIP-induced CAMP formation after pretreatment). Caveolin-1 phosphorylation, and VPAC(2) receptor internalization and desensitization were blocked by disruption of caveolae with M beta 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 VPAC(2) receptor internalization and desensitization. Published by Elsevier Inc.
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