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Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains.

Abstract：

:A number of different agonists activate G protein-coupled receptors to stimulate adenylyl cyclase (AC), increase cAMP formation, and promote relaxation in vascular smooth muscle. To more fully understand this stimulation of AC, we assessed the expression, regulation, and compartmentation of AC isoforms in rat aortic smooth muscle cells (RASMC). Reverse transcription-polymerase chain reaction detected expression of AC3, AC5, and AC6 mRNA, whereas immunoblot analysis indicated expression of AC3 and AC5/6 protein primarily in caveolin-rich membrane (cav) fractions relative to noncaveolin (noncav) fractions. Beta(1)-adrenergic receptors (AR), beta(2)AR, and G(s) were detected in both cav and noncav fractions, whereas the prostanoid receptors EP(2)R and EP(4)R were excluded from cav fractions. We used an adenoviral construct to increase AC6 expression. Overexpressed AC6 localized only in noncav fractions. Two-fold overexpression of AC6 caused enhancement of forskolin-, isoproterenol- and prostaglandin E(2)-stimulated cAMP formation but no changes in basal levels of cAMP. At higher levels of AC6 overexpression, basal and adenosine receptor-stimulated cAMP levels were increased. Stimulation of cAMP levels by agents that increase Ca(2+) in native cells was consistent with the expression of AC3, but overexpression of AC6, which is inhibited by Ca(2+), blunted the Ca(2+)-stimulable cAMP response. These data indicate that: 1) RASMC express multiple AC isoforms that localize in both caveolin-rich and noncaveolin domains, 2) expression of AC6 in non-caveolin-rich membranes can increase basal levels of cAMP and response to several stimulatory agonists, and 3) Ca(2+)-mediated regulation of cAMP formation depends upon expression of different AC isoforms in RASMC. Compartmentation of GPCRs and AC is different in cardiomyocytes than in RASMC, indicating that targeting of these components to caveolin-rich membranes can be cell-specific. Moreover, our results imply that the colocalization of GPCRs and the AC isoforms they activate need not occur in caveolin-rich fractions.

journal_name

Mol Pharmacol

journal_title

Molecular pharmacology

authors

Ostrom RS,Liu X,Head BP,Gregorian C,Seasholtz TM,Insel PA

doi

10.1124/mol.62.5.983

keywords:

subject

Has Abstract

pub_date

2002-11-01 00:00:00

pages

983-92

issue

eissn

0026-895X

issn

1521-0111

journal_volume

pub_type

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Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains.