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.

Cellular organelles and plasma membrane microdomains

Classical descriptions of cellular anatomy define organelles and regions (e.g. nucleus, Golgi apparatus, mitochondria and plasma membrane) as discrete entities. Biochemical efforts to understand the intracellular functions of these entities have primarily involved the isolation and characterization of subcellular fractions. Substantial evidence, however, supports the view that such preparations are quite heterogeneous, with differences in protein and lipid composition as well as in expression of organelle-specific functional activities. This is particularly the case for anatomically distinct regions of the plasma membrane, which can be strikingly non-uniform in expression of molecular components and functional activities. For example, the apical and basolateral membranes of epithelial cells, luminal and abluminal membranes of endothelial cells and dendritic, axonal and cell body membranes of neurons all demonstrate differences in expression of protein and lipid components and in differentiated responses characteristic of those cell types.

Lipid rafts, a subdomain of the plasma membrane, are defined by their enrichment in cholesterol and glycosphingolipids, especially in the outer leaflet of the lipid bilayer, a lipid composition that results in regions of greater order and less fluidity than more disordered portions that have less densely packed phospholipids [1]. Even though they are widely expressed in eukaryotic cells, lipid rafts are flat and cannot be readily visualized microscopically. This difficulty in visualization has contributed to considerable debate regarding the nature and size of lipid rafts; some view their definition, indeed their existence, as operational rather than anatomic [2,3]. In addition to the difficulty of visualizing lipid rafts, the lack of uniformly employed methods to isolate these domains has led to controversy with respect to their expression, properties and functional roles [15]. Nevertheless, the identification of lipid rafts has helped to revise concepts regarding organization of plasma membrane components, in particular to a reformulation of models that hypothesized a lipid ‘sea’ with protein ‘islands’ (e.g. the fluid mosaic model [6]) that required the collision of protein components so as to facilitate biochemical reactions that alter cell function. Identification of lipid raft/caveolae microdomains helped to refine such ideas by providing a basis (i.e. co-localization) to help explain the efficiency of certain biochemical events, such as signal transduction, that occur in the membrane environment and that might otherwise be constrained by the relative inaccessibility of reactants present at low concentrations.

Caveolae as membrane microdomains

Lipid rafts are found in virtually all types of cells, but a number of cell types (with certain important exceptions, e.g. erythrocytes, lymphocytes and neurons) also express caveolae. Caveolae (‘little caves’), first identified microscopically in the 1950s as approx. 100 nm invaginations of the plasma membrane (Figure 1) [7,8], have a lipid composition similar to that of lipid rafts but in addition, possess an organelle-specific, structural protein, caveolin [911]. The discovery of caveolin provided a molecular tag that has facilitated biochemical, cellular and molecular biological studies of caveolar microdomains (for recent reviews, see [1217]). Flotillins/reggies, another class of proteins identified in caveolae, are also found in cells that lack caveolae and in non-caveolar membranes, and thus are probably not as useful as caveolins for such studies [5]. The three known caveolins (caveolin-1, -2 and -3) have a similar overall molecular organization but differ in their primary sequence and expression in particular tissues [13]. Caveolin-3, for example, is uniquely expressed in skeletal and cardiac myocytes. Based on results of biochemical and cell biological studies and from studies of mice in which individual caveolins have been knocked out, the different caveolins have non-identical patterns of regulation of enzymatic and functional activities [13,14].

Localization of caveolae near mitochondria in adult cardiac myocytes

Figure 1
Localization of caveolae near mitochondria in adult cardiac myocytes

Myocytes were fixed in 4% (w/v) paraformaldehdye, 1% glutaraldehyde in 0.1 M cacodylate buffer, for 24 h at 4°C. Sections (70 nm) were stained with uranyl acetate and lead citrate and viewed on Phillips CM10 electron microscope at magnifications of ×21000 (A) and ×5200 (B).

Figure 1
Localization of caveolae near mitochondria in adult cardiac myocytes

Myocytes were fixed in 4% (w/v) paraformaldehdye, 1% glutaraldehyde in 0.1 M cacodylate buffer, for 24 h at 4°C. Sections (70 nm) were stained with uranyl acetate and lead citrate and viewed on Phillips CM10 electron microscope at magnifications of ×21000 (A) and ×5200 (B).

Much effort has been directed at defining the cellular and biochemical activities regulated by lipid rafts and caveolae. These include nutrient transport, endocytosis, exocytosis, transcytosis, viral entry and budding, as well as receptor and ion-channel expression, activation and desensitization [1217]. A monograph summarizing presentations at the Biochemical Society's Annual Symposium ‘BioScience 2004’ provides reviews on a number of such actions, in particular as related to endocytosis and exocytosis [18]. The term ‘caveosome’ has been suggested for a caveolin-containing endosome that mediates endocytosis in a manner parallel to and independent of the endocytic pathway of clathrin-coated pits and clathrin-coated vesicles [3,19]. Glycosylphosphatidylinositol (GPI)-linked proteins and certain others preferentially localize in lipid rafts/caveolae. Shear stress and calcium release are among the cellular responses that appear to be regulated by caveolae. In addition, changes in expression or activity of lipid rafts/caveolae have been implicated in aging and disease-associated alterations in cell function [13,17,18,2023].

Lipid rafts/caveolae and signal transduction

The compartmentation, organization and functional regulation of signal transduction components by lipid rafts and caveolae have attracted considerable interest. Lipid rafts in lymphocytes play a role in cell-activation events, including the regulation of various tyrosine kinases [24,25]. In other cells, caveolae are sites that localize signalling complexes that include GPCRs (G-protein-coupled receptors), heterotrimeric G-proteins and G-protein-regulated effectors. Many physiologically important GPCRs have been localized to lipid raft/caveolae microdomains (Table 1 and [26,27]). In some cases, preassembled signalling complexes localize in those domains but in others, interaction with agonists facilitates migration of GPCR to lipid rafts/caveolae as part of the events involved in desensitization and internalization (Table 1). Results for the angiotensin-1 receptor have implicated a role for GPCR–caveolin interaction as more important for receptor sorting and delivery to the plasma membrane than for localization in membrane caveolae [28]. Other findings describe poorly understood receptor- and cell-specific differences in the contribution of lipid rafts/caveolae to GPCR desensitization and internalization [26]. A portion of cellular G-proteins and G-protein-regulated effector molecules localize along with GPCR to those domains [2937]. Certain GPCRs, G-proteins and effectors show tissue-specific patterns of co-localization in lipid rafts/caveolae; the molecular basis for these patterns is not known [31].

Table 1
Examples of G-protein-coupled receptors that localize in lipid raft/caveolae before (‘pre-agonist’) and/or after (‘post-agonist’) treatment with agonists
Pre-agonistPost-agonist
Endothelin (ETA and ETB) 
Cholecystokinin (CCK)  
Muscarinic cholinergic 
Bradykinin (B1 and B2
Lysophosphatidic acid (LPA-1)  
Angiotensin II (AT-1)  
β1- and β2-adrenergic  
P2Y (P2Y1 
Adenosine A1 
Sphingosine 1-phosphate (EDG-1) 
Smoothened/patched  
Serotonin (5HT2A 
Calcium-sensitive  
α1-Adrenergic (α1B 
Chemokine CCR2  
Metabotropic glutamate (mGlu1)  
Gonadotrophin-releasing hormone (GnRH)  
Oxytocin  
Growth-hormone releasing hormone  
Dopamine [D1; D(1A)] 
Neurokinin 1  
μ-Opioid receptor  
Pre-agonistPost-agonist
Endothelin (ETA and ETB) 
Cholecystokinin (CCK)  
Muscarinic cholinergic 
Bradykinin (B1 and B2
Lysophosphatidic acid (LPA-1)  
Angiotensin II (AT-1)  
β1- and β2-adrenergic  
P2Y (P2Y1 
Adenosine A1 
Sphingosine 1-phosphate (EDG-1) 
Smoothened/patched  
Serotonin (5HT2A 
Calcium-sensitive  
α1-Adrenergic (α1B 
Chemokine CCR2  
Metabotropic glutamate (mGlu1)  
Gonadotrophin-releasing hormone (GnRH)  
Oxytocin  
Growth-hormone releasing hormone  
Dopamine [D1; D(1A)] 
Neurokinin 1  
μ-Opioid receptor  

From a functional point of view, the localization of signalling components in lipid rafts/caveolae can provide a means for rapid, high-fidelity activation in signalling pathways, especially since most such pathways require interaction of multiple proteins. The ‘caveolin/lipid raft signalling hypothesis’ posits that the regulation of signal transduction events occurs as a consequence of interaction of signalling proteins with a ‘caveolin scaffolding domain’ (a 41-amino-acid region in the cytoplasmic N-terminal tail), an interaction that is hypothesized to inhibit such pathways, at least in part, by sequestering components away from signal transduction partners [1214,32].

In GPCR–G-protein-effector systems, all three classes of components localize at the plasma membrane. Their coexpression in caveolae is thus well suited for efficient activation and amplification of GPCR signalling events. Certain AC (adenylyl cyclase) isoforms and G-proteins that regulate AC, Gs and Gi, are among the GPCR pathway components that localize to lipid rafts/caveolae [26,2937]. AC6, a widely expressed AC isoform that localizes in lipid rafts/caveolae in numerous cell types, is susceptible to inhibition by a variety of negative regulators, including protein kinases A and C, RGS2 (regulators of G-protein signalling 2), Gαi, Gβ/γ, Ca2+ and NO. eNOS (endothelial nitric oxide synthase; NOS3), a key source of NO, also localizes to caveolae; this co-localization of eNOS and AC6 appears to contribute to the regulation of cAMP synthesis [34]. Other results implicate a role for lipid rafts/caveolae in ‘receptor transactivation’ between GPCR and tyrosine kinase receptors, such as the EGF (epidermal growth factor) receptor, and in GPCR activation of MAPKs (mitogen-activated protein kinases) [26].

Caveolins in locations other than caveolae: use of microscopy

A growing body of work, including our recent findings, provides evidence for the expression of caveolins in cells independent of caveolae, thus suggesting roles in addition to their structural contribution to caveolae [3,28,35]. Work in cardiac cells has identified functionally important expression of GPCR–G-protein–AC in lipid rafts/caveolae of cardiac myocytes prepared from neonatal rats [2931,34,36,37] but findings with adult rat cardiac myocytes, which are more highly differentiated than neonatal cells, reveal a strikingly different pattern of caveolin-3 expression (Figure 2): a substantial portion of the cells’ caveolin is detected (by sucrose-density-gradient fractionation and immunoprecipitation) in non-buoyant fractions, whereas caveolae, based on their lipid composition, localize to buoyant fractions (4 and 5 in Figure 2). Such findings imply that caveolin is found in domains distinct from caveolae, but do not identify the cellular location of those domains.

Sucrose-density-gradient fractionation of caveolins in adult and neonatal cardiac myocytes (CM) and adult cardiac fibroblasts (CF)

Figure 2
Sucrose-density-gradient fractionation of caveolins in adult and neonatal cardiac myocytes (CM) and adult cardiac fibroblasts (CF)

Cellular membranes were extracted with Na2CO3 in the absence of detergent, fractionated on discontinuous sucrose density gradients and fractions (top to bottom) were immunoblotted for caveolin-1 (Cav-1, CF), caveolin-3 (Cav-3, adult and neonatal CM) and AC5/6, as described in [35,37].

Figure 2
Sucrose-density-gradient fractionation of caveolins in adult and neonatal cardiac myocytes (CM) and adult cardiac fibroblasts (CF)

Cellular membranes were extracted with Na2CO3 in the absence of detergent, fractionated on discontinuous sucrose density gradients and fractions (top to bottom) were immunoblotted for caveolin-1 (Cav-1, CF), caveolin-3 (Cav-3, adult and neonatal CM) and AC5/6, as described in [35,37].

Most approaches that have assessed expression and function of caveolins, especially as related to signalling proteins, have used biochemical methods to infer information regarding subcellular localization. We have recently utilized microscopic techniques, including immunofluoresence with deconvolution analysis as well as immunoelectron microscopy, to examine expression of caveolin and its co-expression with signalling molecules in adult cardiac myocytes [35]. We observed caveolin-3 in intercalated discs (between cells) and in transverse tubule/sarcoplasmic reticulum regions. Similar localization has been observed for caveolin in skeletal myocytes [38]. The findings imply a role for caveolin, most probably independent of caveolae, in the regulation of calcium homoeostasis in cardiac and skeletal muscle [39]. We have also observed close apposition of plasma membrane caveolae and mitochondria in adult cardiac myocytes (Figure 1), findings that suggest ‘communication’ between caveolae and mitochondria.

Some remaining questions regarding lipid rafts/caveolae

The results reviewed here lead to a number of questions, which include: what is the full range of signalling proteins and signalling cascades that are organized in caveolin-rich regions and lipid rafts? The use of proteomic strategies [13,24,40,41] should help answer this question, especially with respect to differences in expression of signalling proteins in lipid rafts and caveolae of different cell types. How equivalent are buoyant fractions that have been isolated by different techniques (e.g. detergent versus non-detergent methods) and how can one distinguish the separate roles of lipid rafts from those of caveolae in such fractions? How accurately do biochemical methods characterize the cellular properties of lipid rafts and caveolae? What are the protein–protein and protein–lipid interactions that determine co-localization and functional regulation of signalling proteins in those microdomains? Do caveolins have similar or different functions in the plasma membrane and other cellular locations? Answers to such questions will probably require development and application of new techniques, such as fluorescence resonance energy transfer of living cells [42,43] and have the potential to dramatically enhance understanding of individual signalling events and signalling networks in cells and to define more precisely the role of lipid rafts and caveolae in such events.

Research Colloquia: Research Colloquia at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by F. Antoni (Edinburgh, U.K.), C. Cooper (Essex, U.K.) and M. Schweizer (Heriot-Watt, U.K.). The first five papers featured in this Section were presented as a part of the Free Radicals and Cyclic Nucleotide Signalling Pathways: Nitric Oxide, Reactive Oxygen Species, cAMP and cGMP Pfizer-Sponsored Research Colloquium.

Abbreviations

     
  • AC

    adenylyl cyclase

  •  
  • eNOS

    endothelial nitric oxide synthase

  •  
  • GPCR

    G-protein-coupled receptor

This work was supported by grants and fellowships from National Institutes of Health (Bethesda, MD, U.S.A.). We thank Marilyn Farquhar and Ingrid Niesman for provision of and assistance with the electron microscope used to generate Figure 1.

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