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

Abstract

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 cellsurface 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, M 4 -muscarinic acetylcholine receptors (mAChR), -opioid receptors, and G␣ s in both BF and HF. In contrast, M 2 -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 -opioid receptors in both sarcolemmal and intracellular membranes, whereas M 2 -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.

Brain region-specific N-glycosylation and lipid rafts association of the rat mu opioid receptor

“…By use of the same non-detergent rafts preparation method employed in this study, we and others have found that mu, delta, or kappa opioid receptors are mainly associated with lipid rafts in several tissues/cells, including rat brain CPu, human placenta, rat cardiac myocytes, NG108-15 cells, HEK293 cells and CHO cells [13,17,20,27,28]. In this regard, the MOR in the rat thalamus is an exception.…”

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

“…Relevant GPCRs and effector molecules also associate with cardiac caveolae/caveolin-3 (Head et al, 2005).…”

Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells

“…Many 7-TMRs/GPCRs have been shown to locate in and regulated by lipid rafts (Pike, 2003;Chini and Parenti, 2004;Ostrom and Insel, 2004), including opioid receptors (Head et al, 2005;Xu et al, 2006;Zhao et al, 2006;Patel et al, 2006;Huang et al, 2007). For example, MOR expressed in HEK293 cells was found by Zhao et al (2006) to be mainly localized in lipid rafts domains of plasma membranes and this localization was required for MOR-mediated adenylyl cyclase superactivation.…”