Caveolin-1 and Lipid Microdomains Regulate Gs Trafficking and Attenuate Gs/Adenylyl Cyclase Signaling (original) (raw)
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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.
British Journal of Pharmacology, 2004
The many components of G-protein-coupled receptor (GPCR) signal transduction provide cells with numerous combinations with which to customize their responses to hormones, neurotransmitters, and pharmacologic agonists. GPCRs function as guanine nucleotide exchange factors for heterotrimeric (a, b, g) G proteins, thereby promoting exchange of GTP for GDP and, in turn, the activation of 'downstream' signaling components. Recent data indicate that individual cells express mRNA for perhaps over 100 different GPCRs (out of a total of nearly a thousand GPCR genes), several different combinations of G-protein subunits, multiple regulators of G-protein signaling proteins (which function as GTPase activating proteins), and various isoforms of downstream effector molecules. The differential expression of such protein combinations allows for modulation of signals that are customized for a specific cell type, perhaps at different states of maturation or differentiation. In addition, in the linear arrangement of molecular interactions involved in a given GPCR-G-proteineffector pathway, one needs to consider the localization of receptors and post-receptor components in subcellular compartments, microdomains, and molecular complexes, and to understand the movement of proteins between these compartments. Co-localization of signaling components, many of which are expressed at low overall concentrations, allows cells to tailor their responses by arranging, or spatially organizing in unique and kinetically favorable ways, the molecules involved in GPCR signal transduction. This review focuses on the role of lipid rafts and a subpopulation of such rafts, caveolae, as a key spatial compartment enriched in components of GPCR signal transduction. Recent data suggest cell-specific patterns for expression of those components in lipid rafts and caveolae. Such domains likely define functionally important, cell-specific regions of signaling by GPCRs and drugs active at those GPCRs.
G-protein coupled receptors in lipid rafts and caveolae: how, when and why do they go there?
Journal of Molecular Endocrinology, 2004
This review describes the advances in our understanding of the role of G-protein coupled receptor (GPCR) localisation in membrane microdomains known as lipid rafts and caveolae. The growing interest in these specialised regions is due to the recognition that they are involved in the regulation of a number of cell functions, including the fine-tuning of various signalling molecules. As a number of GPCRs have been found to be enriched in lipid rafts and/or caveolae by means of different experimental approaches, we first discuss the pitfalls and uncertainties related to the use of these different procedures. We then analyse the addressing signals that drive and/or stabilise GPCRs in lipid rafts and caveolae, and explore the role of rafts/caveolae in regulating GPCR trafficking, particularly in receptor exo-and endocytosis. Finally, we review the growing evidence that lipid rafts and caveolae participate in the regulation of GPCR signalling by affecting both signalling selectivity and coupling efficacy.
Interaction with Caveolin-1 Modulates G Protein Coupling of Mouse 3-Adrenoceptor
Journal of Biological Chemistry, 2012
Background: Caveolins affect signaling by G protein-coupled receptors (GPCRs). Results: Interaction between ! 3a-adrenoceptors and caveolin-1 facilitates Gs-mediated responses but prevents the receptor from coupling to inhibitory Gi/o proteins. Conclusion: Association of the ! 3a-adrenoceptor with caveolin-1 is important in determining the selectivity and efficiency of G protein coupling and signaling. Significance: We demonstrate the functional impact of a GPCR-caveolin association. SUMMARY Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knockout mice suggest that ! 3-AR signaling is dependent on caveolin-1, however it is not known whether caveolin-1 is associated with the ! 3-AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse ! 3a-and ! 3b-AR isoforms that diverge at the distal C-terminus. Only the ! 3b-AR promotes PTX-sensitive cAMP accumulation. When cells expressing the ! 3a-AR were treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, the cyclic AMP response to the ! 3-AR agonist CL316243 became PTX sensitive, suggesting G"i/o coupling. The ! 3a-AR C-terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398) PT, resembles a caveolin interaction motif. Mutant ! 3a-ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type ! 3a-AR but not the mutant receptors. In membrane preparations, the ! 3b-AR activated G"o and mediated PTX-sensitive cAMP responses, whereas the ! 3a-AR did not activate G"i/o proteins. The endogenous ! 3a-AR displayed G"i/o coupling in brown adipocytes from caveolin-1 knockout mice, or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the ! 3a-AR with caveolin inhibits coupling to G"i/o proteins, and suggest that signaling is modulated by a raft-enriched complex containing the ! 3a-AR, caveolin-1, G"s and adenylyl cyclase. The plasma membrane is not a random or uniform array of lipids and proteins, but instead has physical heterogeneity as well as higher order structures that are critical to the functioning of receptors, ion channels and signaling proteins. Membrane rafts, or lipid rafts, are liquid-ordered lipid domains of 5-10 nm that are enriched in cholesterol and sphingolipids (1,2). Rafts display reduced lateral diffusion relative to the liquid-disordered phase, providing nucleation sites for further membrane organization to produce larger structures of 50-150 nm. These higher order structures are enriched in multi-protein complexes, acting as signaling platforms that govern association between receptors and effector proteins (reviewed in (3)). Caveolae represent a subset of
Journal of Biological Chemistry, 2004
Several cell types, including cardiac myocytes and vascular endothelial cells, produce nitric oxide (NO) via both constitutive and inducible isoforms of NO synthase. NO attenuates cardiac contractility and contributes to contractile dysfunction in heart failure, although the precise molecular mechanisms for these effects are poorly defined. Adenylyl cyclase (AC) isoforms type 5 and 6, which are preferentially expressed in cardiac myocytes, may be inhibited via a direct nitrosylation by NO. Because endothelial NO synthase (eNOS and NOS3), -adrenergic (AR) receptors, and AC6 all can localize in lipid raft/caveolin-rich microdomains, we sought to understand the role of lipid rafts in organizing components of AR-G s -AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with -cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. ARand G s -mediated activation of AC activity were inhibited by -cyclodextrin treatment, but prostanoid receptor-stimulated AC activity, which appears to occur outside caveolin-rich microdomains, was unaffected unless eNOS was overexpressed and lipid rafts were disrupted. An NO donor, SNAP, inhibited basal and forskolin-stimulated cAMP production in both native cardiac myocytes and cardiac myocytes and pulmonary artery endothelial cells engineered to overexpress AC6. These effects of SNAP were independent of guanylyl cyclase activity and were mimicked by overexpression of eNOS. The juxtaposition of eNOS with AR and AC types 5 and 6 results in selective regulation of AR by eNOS activity in lipid raft domains over other G s -coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity. pulmonary artery endothelial cell(s).
Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease
Biochemistry and Cell Biology-biochimie Et Biologie Cellulaire, 2004
Caveolae were initially described some 50 years ago. For many decades, they remained predominantly of interest to structural biologists. The identification of a molecular marker for these domains, caveolin, combined with the possibility to isolate such cholesterol-and sphingolipid-rich regions as detergent-insoluble membrane complexes paved the way to more rigorous characterization of composition, regulation, and function. Experiments with knock-out mice for the caveolin genes clearly demonstrate the importance of caveolin-1 and -3 in formation of caveolae. Nonetheless, detergent-insoluble domains are also found in cells lacking caveolin expression and are referred to here as lipid rafts. Caveolae and lipid rafts were shown to represent membrane compartments enriched in a large number of signaling molecules whose structural integrity is essential for many signaling processes. Caveolin-1 is an essential structural component of cell surface caveolae, important for regulating trafficking and mobility of these vesicles. In addition, caveolin-1 is found at many other intracellular locations. Variations in subcellular localization are paralleled by a plethora of ascribed functions for this protein. Here, more recent data addressing the role of caveolin-1 in cellular signaling and the development of diseases like cancer will be preferentially discussed.
Cancer Science, 2007
Caveolin-1 is a component of lipid rafts, and is considered to be a tumor suppressor molecule. However, the mechanisms by which caveolin-1 functions in cancer cells are not well understood. We generated caveolin-1 transfectant cells (Cav-1 + cells) using a human melanoma cell line (SK-MEL-28) and investigated the effects of caveolin-1 overexpression on the GD3-mediated malignant properties of melanomas. Cav-1 + cells had decreased cell growth and motility, and reduced phosphorylation levels of p130Cas and paxillin relative to controls. In floatation analysis, although GD3 was mainly localized in glycolipid-enriched microdomain (GEM)/rafts in control cells, it was dispersed from GEM/rafts in Cav-1 + cells. Correspondingly, GD3 in Cav-1 + cells stained uniformly throughout the membrane, whereas control cells showed partial staining of the membrane, probably at the leading edge. p130Cas and paxillin were stained in the leading edges and colocalized with GD3 in the control cells. In contrast, these molecules were diffusely stained and no definite leading edges were detected in Cav-1 + cells. These results suggest that caveolin-1 regulates GD3-mediated malignant signals by altering GD3 distribution and leading edge formation. These results reveal one of the mechanisms by which caveolin-1 curtails the malignant properties of tumor cells. (Cancer Sci 2007; 98: 512-520)
Glia, 2005
Caveolae represent membrane microdomains acting as integrators of cellular signaling and functional processes. Caveolins are involved in the biogenesis of caveolae and regulate the activity of caveolae-associated proteins. Although caveolin proteins are found in the CNS, the regulation of caveolins in neural cells is poorly described. In the present study, we investigated different modes and mechanisms of caveolin gene regulation in primary rat astrocytes. We demonstrated that activation of cAMP-dependent signaling pathways led to a marked reduction in protein levels of caveolin-1/-2 in cortical astrocytes. Application of transforming growth factor-␣ (TGF-␣) also resulted in a decrease of caveolin-1/-2 expression. Decreased caveolin protein levels were mirrored by diminished caveolin gene transcription. The repressive effect of TGF-␣ on caveolin-1 expression was MAP kinase-independent and partly mediated through the PI3-kinase pathway. Further downstream, inhibition of histone deacetylases abrogated TGF-␣ effects, suggesting that chromatin remodeling processes could contribute to caveolin-1 repression. Intriguingly, alterations of caveolin gene expression in response to cAMP or TGF-␣ coincided with reciprocal and brain-region specific changes in glial glutamate transporter GLT-1 expression. The reciprocal regulation of caveolin-1 and GLT-1 expression might be gated through a common PI3-kinase dependent pathway triggered by TGF-␣. Finally, we showed that GLT-1 is located in non-caveolar lipid rafts of cortical astrocytes. In conclusion, this study highlights the occurrence of the reciprocal regulation of caveolin and GLT-1 expression during processes such as astrocyte differentiation via common signaling pathways. We also provide strong evidence that GLT-1 itself is concentrated in lipid rafts, inferring an important role for glial glutamate transporter function.
Caveolae and Lipid Rafts: G Protein-Coupled Receptor Signaling Microdomains in Cardiac Myocytes
Annals of The New York Academy of Sciences, 2005
A BSTRACT : A growing body of data indicates that multiple signal transduction events in the heart occur via plasma membrane receptors located in signaling microdomains. Lipid rafts, enriched in cholesterol and sphingolipids, form one such microdomain along with a subset of lipid rafts, caveolae, enriched in the protein caveolin. In the heart, a key caveolin is caveolin-3, whose scaffolding domain is thought to serve as an anchor for other proteins. In spite of the original morphologic definition of caveolae (" little caves"), most work related to their role in compartmenting signal transduction molecules has involved subcellular fractionation or immunoprecipitation with anti-caveolin antibodies. Use of such approaches has documented that several G protein-coupled receptors (GPCR), and their cognate heterotrimeric G proteins and effectors, localize to lipid rafts/caveolae in neonatal cardiac myocytes. Our recent findings support the view that adult cardiac myocytes appear to have different patterns of localization of such components compared to neonatal myocytes and cardiac fibroblasts. Such results imply the existence of multiple subcellular microdomains for GPCR-mediated signal transduction in cardiac myocytes, in particular adult myocytes, and raise a major unanswered question: what are the precise mechanism(s) that determine co-localization of GPCR and post-receptor components with lipid rafts/caveolae in cardiac myocytes and other cell types? K EYWORDS : adenylyl cyclase; cardiac fibroblast; cardiac myocyte; caveolin; G proteins; GPCR
Journal of Biological Chemistry, 2001
The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.