Abstract. Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation.We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue-an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain *85 % of caveolin and ,',,55 % of a GPI-linked marker protein, while they exclude I>98% of integral plasma membrane protein markers and t>99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules-including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap I-the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.

Abstract. GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPIlinked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8 % of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organellespecific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmicaUy-oriented signaling molecules, including: (a) GTP-binding proteins-both small and heterotrimeric; (b) annexin N-an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes-an apically localized member of the Src family of non-receptor type protein-tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrahe adaptor molecule that couples luminal GPIlinked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPImediated signal transduction events. In addition, our results have implications for understanding v-Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin interacts directly with heterotrimeric G-proteins and can functionally regulate their activity. Recently, a second caveolin gene has been identified and termed caveolin-2. Here, we report the molecular cloning and expression of a third member of the caveolin gene gamily, caveolin-3. Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are approximately 65% identical and approximately 85% similar. A single stretch of eight amino acids (FED-VIAEP) is identical in caveolin-1, -2, and -3. This conserved region may represent a "caveolin signature sequence" that is characteristic of members of the caveolin gene family. Caveolin-3 mRNA is expressed predominantly in muscle tissue-types (skeletal muscle, diaphragm, and heart) and is selectively induced during the differentiation of skeletal C2C12 myoblasts in culture. In many respects, caveolin-3 is similar to caveolin-1: (i) caveolin-3 migrates in velocity gradients as a high molecular mass complex; (ii) caveolin-3 colocalizes with caveolin-1 by immunofluorescence microscopy and cell fractionation studies; and (iii) a caveolin-3-derived polypeptide functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins. Identification of a muscle-specific member of the caveolin gene family may have implications for understanding the role of caveolin in different muscle cell types (smooth, cardiac, and skeletal) as previous morphological studies have demonstrated that caveolae are abundant in these cells. Our results also suggest that other as yet unknown caveolin family members are likely to exist and may be expressed in a regulated or tissue-specific fashion.

A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization ofthese purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as fTishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.Caveolae are plasma membrane specializations (1). Caveolin, a 21-to 24-kDa integral membrane protein, has been identified as a principal component of caveolae membranes in vivo (2, 3). Purification of caveolin-rich membrane domains reveals several distinct classes of signaling molecules (3-6). These include heterotrimeric guanine nucleotide binding proteins (G proteins; a and 13'y subunits), Src-like kinases, protein kinase Ca, and Rap GTPases. Based on these observations, we have proposed (1) that caveolin may function as a scaffolding protein to organize and concentrate inactive signaling molecules within caveolae membranes-for activation by appropriate receptors. This caveolae signaling hypothesis states that "compartmentalization of ceftain cytoplasmic signaling molecules within caveolae could allow efficient and rapid coupling of activated receptors to more than one effector system" (1). In support of this view, inactive G a subunits interact directly with caveolin in a 1:1 stoichiometry-holding them in an inactive conformation (7). Thus, knowledge of the subunit structure of caveolin is important for understanding how caveolin might function to organize or concentrate G a subunits and other signaling molecules within caveolae membranes.In this report, we show that caveolin interacts with itself to form a discrete high molecular mass oligomer. As caveolin also interacts with G a subunits, self oligomerization of caveolin could provide a means for concentrating trimeric G proteins and other caveolin-interacting molecules within caveolae. The existence of caveolin homooligomers complexed with inactive G proteins could explain the observations of Rodbell and colleagues (8), who observed that inactive G proteins exist as high molecular mass oligomeric complexes and that activated G proteins dissociate to monomers. Similarly, activated G proteins fail to interact with recombinant caveolin (7). MATERIALS & METHODSMaterials. Antibodies to carbonic anhydrase IV and glutathione S-transferase (GST) were gifts of W. S. Sly (St. Louis University) and R. Young (Whitehead I...

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