Caveolae and Lipid Rafts: G Protein-Coupled Receptor Signaling Microdomains in Cardiac Myocytes (original) (raw)
Related papers
Journal of Biological Chemistry, 2005
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.
Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte
Journal of Molecular and Cellular Cardiology, 2008
Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing receptor-specific signals to be generated by a single second messenger. Here we determine whether caveolae, invaginated lipid rafts, are responsible for confining the β 2 adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MβC) to disrupt caveolae. Caveolae-containing membrane fractions were isolated by detergent-free sucrose gradient fractionation. Cell shortening and phosphorylation of the sarcoplasmic reticular protein phospholamban (PLB) and the myofilament protein troponin I (TnI) were measured in response to β 2 AR stimulation (with salbutamol in the presence of 1 μM atenolol). Ser 16 phosphorylation of PLB (pPLB), Ser 22,23 phosphorylation of TnI (pTnI), and positive lusitropy were used as indices of global cAMP signals. The ability of MβC to disrupt caveolae was confirmed by selective depletion of the buoyant membrane fractions of cholesterol and caveolin 3, the 2 essential components of caveolae. In control cells, no change in pPLB, pTnI or time to half relaxation was recorded with β 2 AR stimulation (P N 0.05), but following caveolar disruption a 60-70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P b 0.05). These data show for the first time that disrupting caveolae converts the sarcolemmal-confined cAMP signal associated with β 2 AR stimulation to a global signal that targets proteins of the sarcoplasmic reticulum and myofilaments, with functional sequelae. The role of caveolae in spatial control of cAMP may be relevant to perturbation of β AR signalling in cardiovascular disease.
Biochemical Society Transactions, 2005
G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.
Journal of Cellular Biochemistry, 2000
Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse ␣and -subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.
1Adrenergic receptor signaling is localized to caveolae in neonatal rat cardiomyocytes
Journal of Molecular and Cellular Cardiology, 2006
In neonatal rat cardiomyocytes, phosphatidylinositol(4,5)bisphosphate (PIP 2 ) is a precursor of second messengers, a stabilizer of ion channels and exchangers, an anchor point for the cytoskeleton and, in addition, can serve as a signaling molecule in its own right. We examined the possibility that sarcolemmal PIP 2 exists in different pools and that only one of these provides the substrate for α 1 -adrenergic receptor activated phospholipase C (PLC). Membranes were separated on the basis of buoyant density, and the light lipid raft fractions were further separated into caveolae and noncaveolar rafts using immunoprecipitation. PIP 2 was principally located in the light lipid raft fractions and was equally distributed between caveolae and non-caveolar membranes. Heavier membrane fractions also contained some PIP 2 . Addition of the α 1 -adrenergic receptor agonist phenylephrine (50 μM) caused reductions in PIP 2 , but only in caveolae. PIP 2 in other fractions was unaffected. In agreement with this, PLCβ1 and, to a lesser extent, Gαq were concentrated in this fraction. PLCβ3 was primarily observed in heavier membranes. We conclude that PIP 2 in cardiomyocyte sarcolemma is compartmentalized and that α 1 -adrenergic receptor signaling is localized to caveolae.
Myocardin Family Members Drive Formation of Caveolae
PLOS ONE, 2015
Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.
Cardiovascular Research, 2001
Objective: Caveolin, a major protein component of caveolae, is now considered to be an inhibitor of cellular growth and proliferation. In this study, we examined the localization of the molecules involved in a1-adrenergic receptor signal relative to that of caveolin in the heart and the changes in caveolin expression during the development of hypertrophy in SHR. Methods: We purified the caveolar protein fractions from rat cardiac tissues, H9C2 cells, and rat vascular smooth muscle cells. Using radioligand receptor binding assay and immunoblot analysis, we examined the distribution and the amount of a1-AR and caveolin. Results: Caveolin-3, the a1-adrenergic receptor, Gq and PLC-b ubtypes (PLC-b1,-b3) were found exclusively in the caveolar fraction in the above tissues. Caveolin-3 were co-immunoprecipitated with a1-adrenergic receptor and Gq from the cardiac tissues. The amount of caveolin subtypes expression (caveolin-1 and-3) and the amount of the a1-adrenergic receptor were examined in the hearts of SHR and age-matched WKY (4-and 24-weeks-old). The amount of caveolin-3 expression was significantly smaller in SHR at 24-weeks-old than that in SHR at 4-weeks-old and that in WKY at 24-weeks-old. Conclusions: The molecules involved in a1-adrenergic signaling are confined to the same microdomain as caveolin. A decrease in caveolin-3 expression may play a role in the development of cardiac hypertrophy in SHR, presumably through de-regulating the inhibition of growth signal in the hearts of SHR in the hypertrophic stage.
Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts
AJP: Heart and Circulatory Physiology, 2008
Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling, and facilitates free fatty acid (FA) uptake by interacting with several fatty acid transport proteins. We previously reported that Cav3 knockout mice (Cav3ko) develop cardiac hypertrophy with diminished contractile function, however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that uptake and oxidation of fatty acids and glucose were normal in hypertrophic Cav3ko hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including fatty acid translocase (CD36) and carnitine palmitoyl transferase (CPT-1) in Cav3ko hearts. Interestingly, myocardial cAMP content was significantly increased by 42%, however, this had no affect on PKA activity in Cav3ko hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a 4-fold increase in the expression of cellular retinol binding protein-III and 3.5-fold increase in 17 -hydroxysteroid dehydrogenase type11 (17β-HSD11), a member of the short-chain dehydrogenase/reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. exercise training exhibits different gene expression of enzymes in energy metabolism. Hypertens Res 26: 829-837, 2003. 30. Lang CH, Bagby GJ, Buday AZ, and Spitzer JJ. The contribution of gluconeogenesis to glycogen repletion during glucose infusion in endotoxemia. Metabolism 36: 180-187, 1987. 31. Lissandron V, and Zaccolo M. Compartmentalized cAMP/PKA signalling regulates cardiac excitation-contraction coupling. J Muscle Res Cell Motil 27: 399-403, 2006. 32. Luiken JJ, Willems J, Coort SL, Coumans WA, Bonen A, Van Der Vusse GJ, and Glatz JF. Effects of cAMP modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes. Biochem J 367: 881-887, 2002. 33. MCMAHON EG, WENNING Q, GOELLNER J, and RUDOLPH AE. CARDIAC-SPECIFIC 11 BETA HYDROXYSTEROID DEHYDROGENASE TYPE 2 TRANSGENIC MICE. edited by CORP P. US: 2004. 34. Kubota I. Fatty acid metabolism assessed by 125I-iodophenyl 9-methylpentadecanoic acid (9MPA) and expression of fatty acid utilization enzymes in volume-overloaded hearts. Eur J Clin Invest 34: 176-181, 2004. 36. Movsesian MA, and Bristow MR. Alterations in cAMP-mediated signaling and their role in the pathophysiology of dilated cardiomyopathy. Curr Top Dev Biol 68: 25-48, 2005. 37. Nielsen LB, Veniant M, Boren J, Raabe M, Wong JS, Tam C, Flynn L, Vanni-Reyes T, Gunn MD, Goldberg IJ, Hamilton RL, and Young SG. Genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heart: evidence that the heart has the capacity to synthesize and secrete lipoproteins. Circulation 98: 13-16, 1998. 38. Odley A, Hahn HS, Lynch RA, Marreez Y, Osinska H, Robbins J, and Dorn GW, 2nd. Regulation of cardiac contractility by Rab4-modulated beta2-adrenergic receptor recycling. Proc Natl Acad Sci U S A 101: 7082-7087, 2004. 39. Park TS, Yamashita H, Blaner WS, and Goldberg IJ. Lipids in the heart: a source of fuel and a source of toxins. Curr Opin Lipidol 18: 277-282, 2007. 40. Parton RG, Way M, Zorzi N, and Stang E. Caveolin-3 associates with developing T-tubules during muscle differentiation. J Cell Biol 136: 137-154, 1997. 41. Ratajczak P, Oliviero P, Marotte F, Kolar F, Ostadal B, and Samuel JL. Expression and localization of caveolins during postnatal development in rat heart: implication of thyroid hormone. J Appl Physiol 99: 244-251, 2005. 42.
Histochemistry and Cell Biology, 2012
Caveolae and caveolins, structural components of caveolae, are associated with specific ion channels in cardiac myocytes. We have previously shown that P2X purinoceptor 7 (P2X7R), a ligand gated ion channel, is increased in atrial cardiomyocytes of caveolin-1 knockout mice; however, the specific biochemical relationship of P2X7R with caveolins in the heart is not clear. The aim of this work was to study the presence of the P2X7R in atrial cardiomyocytes and its biochemical relationship to caveolin-1 and caveolin-3. Caveolin isoforms and P2X7R were predominantly localized in buoyant membrane fractions (lipid rafts/caveolae) prepared from hearts using detergent-free sucrose gradient centrifugation. Caveolin-1 knockout mice showed normal distribution of caveolin-3 and P2X7R to byoyant membranes indicating the importance of caveolin-3 to formation of caveolae. Using clear native PAGE, we showed that caveolin-1, -3 and P2X7R contribute to the same protein complex in membranes of heart atrial cardiomyocytes and in the immortal cardiomyocyte cell line HL-1. Western blot analysis revealed increased caveolin-1 and -3 protein in tissue homogenates of P2X7R knockout mice. Finally, tissue homogenates of atrial tissues from caveolin-3 knockout mice showed elevated mRNA for P2X7R in atria. The colocalization of caveolins with P2X7R in a biochemical complex and compensated upregulation of P2X7R or caveolins in the absence of any component of the complex suggests P2XR7 and caveolins may serve an important regulatory control point for disease pathology in the heart.
2002
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.  1-Adrenergic receptors (AR),  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. Twofold overexpression of AC6 caused enhancement of forskolin-, isoproterenol-and prostaglandin E 2stimulated 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 2ϩ in native cells was consistent with the expression of AC3, but overexpression of AC6, which is inhibited by Ca 2ϩ , blunted the Ca 2ϩ-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 2ϩ-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.