Michael P. Lisanti

Caveolins, a Family of Scaffolding Proteins for Organizing “Preassembled Signaling Complexes” at the Plasma Membrane

Caveolae are vesicular invaginations of the plasma membrane. The chief structural proteins of caveolae are the caveolins. Caveolins form a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules. Because the responsibilities assigned to caveolae continue to increase, this review will focus on: (i) caveolin structure/function and (ii) caveolae-associated signal transduction. Studies that link caveolae to human diseases will also be considered.

Caveolins, Liquid-Ordered Domains, and Signal Transduction

Caveolae were originally identified as flask-shaped invaginations of the plasma membrane in endothelial and epithelial cells (14). Prior to the development of biochemical methods for their purification, caveolae were thought to principally mediate the transcellular movement of molecules (101, 145). Recently, the development of novel purification procedures has greatly expanded our knowledge regarding the putative functions of caveolae in vivo. In this review, we seek to update the working definition of caveolae, describe the functional roles of the caveolin gene family, and summarize the evidence that supports a role for caveolae as mediators of a number of cellular signaling processes.

Caveolae: From Cell Biology to Animal Physiology

Among the membrane compartments of a cell, vesicles known as “caveolae” have long defied functional characterization. However, since the identification of a family of proteins termed “caveolins”, that form and reside in caveolae, a better understanding has emerged. It is now clear that caveolae do not merely play a singular role in the cell, but are pleiotropic in nature—serving to modulate many cellular functions. The purpose of this review is to explicate what is known about caveolins/caveolae and highlight growing areas of caveolar research.

Identification of Peptide and Protein Ligands for the Caveolin-scaffolding Domain IMPLICATIONS FOR THE INTERACTION OF CAVEOLIN WITH CAVEOLAE-ASSOCIATED PROTEINS

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein α subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolin-scaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules. Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries. These caveolin-selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases. A known caveolin-interacting protein, Gi2α, was used as a ligand to further investigate the nature of this interaction. Gi2α and other G-protein α subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from Gi2α interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of Gi2α. This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding domain. In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands. Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions. These results serve to further demonstrate the specificity of this interaction. The implications of our current findings are discussed regarding other caveolin- and caveolae-associated proteins.

Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo.

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathioneS-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61–101) and to a lesser extent the C-terminal tail (amino acids 135–178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

Src Tyrosine Kinases, Gα Subunits, and H-Ras Share a Common Membrane-anchored Scaffolding Protein, Caveolin CAVEOLIN BINDING NEGATIVELY REGULATES THE AUTO-ACTIVATION OF Src TYROSINE KINASES

Caveolae are plasma membrane specializations present in most cell types. Caveolin, a 22-kDa integral membrane protein, is a principal structural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signaling molecules, including Gα subunits, H-Ras, c-Src, and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with Gα subunits and H-Ras, preferentially recognizing the inactive conformation of these molecules. However, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. Here, we examine the structural and functional interaction of caveolin with Src family tyrosine kinases. Caveolin was recombinantly expressed as a glutathione S-transferase fusion. Using an established in vitro binding assay, we find that caveolin interacts with wild-type Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src). Thus, it appears that caveolin prefers the inactive conformation of Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase and Fyn, a related Src family tyrosine kinase. We further analyzed the effect of caveolin on c-Src activity in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosine kinase activity of c-Src as measured via an immune complex kinase assay. Thus, it appears that caveolin structurally and functionally interacts with wild-type c-Src via caveolin residues 82-101. Besides interacting with Src family kinases, this cytosolic caveolin domain (residues 82-101) has the following unique features. First, it is required to form multivalent homo-oligomers of caveolin. Second, it interacts with G-protein α-subunits and down-regulates their GTPase activity. Third, it binds to wild-type H-Ras. Fourth, it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions. Thus, we have termed this 20-amino acid stretch of caveolin residues the caveolin scaffolding domain.

Interaction of a Receptor Tyrosine Kinase, EGF-R, with Caveolins CAVEOLIN BINDING NEGATIVELY REGULATES TYROSINE AND SERINE/THREONINE KINASE ACTIVITIES

Caveolin, a 21–24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein α subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinasein vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.

Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis

Caveolae, 50-100 nm invaginations that represent a subcompartment of the plasma membrane, have been known for many years, but their exact roles remain uncertain. The findings that the caveolae coat protein caveolin is a v-Src substrate and that G-protein-coupled receptors are present in caveolae have suggested a relationship between caveolae, caveolin and transmembrane signalling. The recent isolation of caveolin-rich membrane domains in which caveolin exists as a hetero-oligomeric complex with integral membrane proteins and known cytoplasmic signalling molecules provides support for this hypothesis. Compartmentalization of certain signalling molecules within caveolae could allow efficient and rapid coupling of activated receptors to more than one effector system.

Emerging Themes in Lipid Rafts and Caveolae

We would especially like to thank the following meeting participants who generously contributed to the organization and writing of this meeting review: Miguel Alonso, Toyoshi Fujimoto, Jean Gruenberg, Hai-Tao He, Vaclav Horejsi, Akihiro Kusumi, Tony Magee, Fred Maxfield, Satyajit Mayor, Mark McNiven, Roger Morris, Robert Parton, Radu-Virgil Stan, Claudia Stuermer, and Gisou van der Goot. M.P.L would like to thank Dr. Federica Sotgia for her support and inspiration.M.P.L. is supported by grants from the National Institutes of Health (NIH), the Muscular Dystrophy Association (MDA), the American Heart Association (AHA), and the Komen Breast Cancer Foundation, as well as a Hirschl/Weil-Caulier Career Scientist Award. F.G. is the recipient of a Scientist Development Grant from the American Heart Association (AHA). B.R. is supported by a National Institutes of Health Medical Scientist Training Grant (T32-GM07288).

Differential Targeting of β-Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae A MECHANISM TO FUNCTIONALLY REGULATE THE cAMP SIGNALING PATHWAY

Differential modes for β1- and β2-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in β2-ARs, Gαi, protein kinase A RIIα subunits, caveolin-3, and flotillins (caveolin functional homologues); β1-ARs, m2-muscarinic cholinergic receptors, Gαs, and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIα subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface β2-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different β2-AR expression levels), indicating that the fidelity of β2-AR targeting to caveolae is maintained over a physiologic range of β2-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of β2-ARs (but not β1-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the β-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.