G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes.
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); beta2-adrenergic receptors (AR) in BF; and AC5/6, beta1-AR, M4-muscarinic acetylcholine receptors (mAChR), mu-opioid receptors, and Galpha(s) in both BF and HF. In contrast, M2-mAChR, Galpha(i3), and Galpha(i2) were found only in HF. Immunofluorescence microscopy showed co-localization of Cav-3 with AC5/6, Galpha(s), beta2-AR, and mu-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 Galpha(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.
Head
Brian P
BP
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA.
Patel
Hemal H
HH
Roth
David M
DM
Lai
N Chin
NC
Niesman
Ingrid R
IR
Farquhar
Marilyn G
MG
Insel
Paul A
PA
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D051244:Caveolin 3; D022461:Caveolins / Q000378:metabolism*; D002458:Cell Fractionation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D043585:Receptor, Muscarinic M2 / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism; D011957:Receptors, Opioid / Q000378:metabolism; D012508:Sarcolemma / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction / Q000502:physiology*
D015415:Biomarkers; C495549:Cav3 protein, rat; D051244:Caveolin 3; D022461:Caveolins; D044385:GTP-Binding Protein alpha Subunits; D007527:Isoenzymes; D043585:Receptor, Muscarinic M2; D011943:Receptors, Adrenergic, beta; D043562:Receptors, G-Protein-Coupled; D011976:Receptors, Muscarinic; D011957:Receptors, Opioid; D000242:Cyclic AMP; D000262:Adenylyl Cyclases