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J. Biol. Chem., Vol. 280, Issue 35, 31036-31044, September 2, 2005

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G-protein-coupled Receptor Signaling Components Localize in Both Sarcolemmal and Intracellular Caveolin-3-associated Microdomains in Adult Cardiac Myocytes^*

Brian P. Head, Hemal H. Patel, David M. Roth¶, N. Chin Lai||, Ingrid R. Niesman**, Marilyn G. Farquhar**, and Paul A. Insel

From the Departments of Pharmacology, ^¶Anesthesiology, ^||Medicine, and ^**Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0636 and the Veterans Affairs San Diego Healthcare System, San Diego, California 92161

This study tests the hypothesis that G-protein-coupled receptor (GPCR) signaling components involved in the regulation of adenylyl cyclase (AC) localize with caveolin (Cav), a protein marker for caveolae, in both cell-surface and intracellular membrane regions. Using sucrose density fractionation of adult cardiac myocytes, we detected Cav-3 in both buoyant membrane fractions (BF) and heavy/non-buoyant fractions (HF); ₂-adrenergic receptors (AR) in BF; and AC5/6, ₁-AR, M₄-muscarinic acetylcholine receptors (mAChR), µ-opioid receptors, and G_s in both BF and HF. In contrast, M₂-mAChR, G_i3, and G_i2 were found only in HF. Immunofluorescence microscopy showed co-localization of Cav-3 with AC5/6, G_s, ₂-AR, and µ-opioid receptors in both sarcolemmal and intracellular membranes, whereas M₂-mAChR were detected only intracellularly. Immunofluorescence of adult heart revealed a distribution of Cav-3 identical to that in isolated adult cardiac myocytes. Upon immunoelectron microscopy, Cav-3 co-localized with AC5/6 and G_s in sarcolemmal and intracellular vesicles, the latter closely allied with T-tubules. Cav-3 immunoprecipitates possessed components that were necessary and sufficient for GPCR agonist-promoted stimulation and inhibition of cAMP formation. The distribution of GPCR, G-proteins, and AC with Cav-3 in both sarcolemmal and intracellular T-tubule-associated regions indicates the existence of multiple Cav-3-localized cellular microdomains for signaling by hormones and drugs in the heart.

Received for publication, March 8, 2005 , and in revised form, June 14, 2005.

^* This work was supported by National Institutes of Health Grants HL66941, HL53773, HL63885, HL074625, and RGS CA1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of California, San Diego, 9500 Gilman Dr., BSB 3076, La Jolla, CA 92093-0636. Tel.: 858-534-2295; Fax: 858-822-1007; E-mail: pinsel{at}ucsd.edu.

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