Massimo Sargiacomo

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.

Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G alpha subunits prevents the interaction of caveolin with G proteins, indicating that inactive G alpha subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

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 GPI-linked 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 organelle-specific 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 cytoplasmically-oriented signaling molecules, including: (a) GTP-binding proteins--both small and heterotrimeric; (b) annex II--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 transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated 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.

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 approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 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 1--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.

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. 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 are vesicular organelles that represent a subdivision of the plasma membrane. They are most abundant in terminally differentiated cell types, i.e. adipocytes, endothelial cells, and muscle cells (skeletal, cardiac, and smooth), although they are found in most cells (1Couet J. Li S. Okamoto T. Sherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar, 2Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (589) Google Scholar, 3Anderson R.G.W. Curr. Opin. Cell Biol. 1993; 5: 647-652Crossref PubMed Scopus (171) Google Scholar). It has been suggested that caveolae may function as subcellular compartments to (i) store inactive signaling molecules for regulated activation and (ii) to facilitate cross-talk between distinct signaling cascades (1Couet J. Li S. Okamoto T. Sherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar, 2Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (589) Google Scholar, 3Anderson R.G.W. Curr. Opin. Cell Biol. 1993; 5: 647-652Crossref PubMed Scopus (171) Google Scholar, 4Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (862) Google Scholar). Caveolin, a 21–24-kDa integral membrane protein, is a major structural component of caveolae. Several independent lines of evidence indicate that caveolin may function as a scaffolding protein within caveolae membranes. (i) Both the NH2-terminal and COOH-terminal domains of caveolin face the cytoplasm, allowing them to freely interact with cytosolic molecules (5Dupree P. Parton R.G. Raposo G. Kurzchalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar, 6Dietzen D.J. Hastings W.R. Lublin D.M. J. Biol. Chem. 1995; 270: 6838-6842Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 7Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). (ii) Caveolin undergoes two stages of oligomerization. First, caveolin monomers assemble into discrete multivalent oligomers containing ∼14–16 monomers per oligomer (8Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (477) Google Scholar, 9Monier S. Parton R.G. Vogel F. Behlke J. Henske A. Kurzchalia T. Mol. Biol. Cell. 1995; 6: 911-927Crossref PubMed Scopus (401) Google Scholar). Subsequently, these individual caveolin homo-oligomers (4–6-nm particles) can interact with each other to form caveolae-like structures in vitro (25–50-nm clusters) (8Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (477) Google Scholar). (iii) Interaction of caveolin with purified heterotrimeric G-proteins and c-Src functionally suppresses their enzymatic activity, holding these proteins in an inactive conformation (10Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 11Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). Thus, caveolin may organize the formation of caveolae microdomains and regulate caveolae-related signaling events. Using a variety of domain mapping approaches (deletion mutagenesis, GST fusion proteins, synthetic peptides), a region within caveolin has been defined that mediates the interaction of caveolin with itself and other proteins. This cytoplasmic 41-amino acid membrane-proximal region of caveolin is sufficient to mediate the formation of caveolin homo-oligomers (8Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (477) Google Scholar), and the carboxyl-terminal half of this region (20 amino acids, residues 82–101) mediates the interaction of caveolin with G-protein α subunits and Src-family tyrosine kinases (10Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 11Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). This caveolin region preferentially recognizes the inactive conformation of these molecules, as mutationally activated Gα subunits (Gs, Q227L), v-Src, and H-Ras to interact with caveolin (10Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 11Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, K.S. Li S. Okamoto T. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). this caveolin domain 82–101) is for caveolin and the interaction of caveolin with certain caveolae-associated proteins eNOS, and Src-family we have previously termed this protein domain the caveolin scaffolding domain S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google G. P. Couet Li S. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is the of these the caveolin scaffolding domain recognizes a motif within caveolin binding signaling this we have the caveolin scaffolding domain as a receptor to caveolin binding peptide peptide the of display Two caveolin binding motifs and amino or were and these motifs within most caveolae-associated proteins J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, caveolin binding motifs mediate the interaction of proteins with the scaffolding domain of caveolin Caveolin binding motifs are present within most and within the kinase domains of distinct of protein kinases protein kinase growth growth receptor receptor kinase cytoplasmic protein kinase growth growth receptor receptor kinase cytoplasmic kinases protein tyrosine kinases and receptor tyrosine kinases the of Src-family kinases, it caveolin with these kinases or can functionally regulate their Here, we present evidence that the EGF-R kinase with the caveolin scaffolding domain a conserved caveolin binding motif that is within the kinase domain of most receptor tyrosine kinases. We also show that caveolin binding can functionally regulate the kinase activity of EGF-R in vitro. Thus, EGF-R is the signaling receptor to be shown to interact with In of our receptor tyrosine kinases as well as their signaling cascades have been previously to caveolae or caveolae-related EGF-R, growth factor receptor, and growth factor receptor P. R.G.W. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, R.G.W. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (403) Google Scholar, J. Cell Biol. 1995; PubMed Scopus Google Scholar, P.E. J. J. Cell Biol. 1996; Google Scholar, S. R.G.W. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). and their were as 1 and 1 of the form of the receptor and variety of other were purified EGF-R kinase residues of the and protein by the of and by The of protein kinase and were cells and cells were in with and were for were two with and in for membrane were as previously K.S. Li S. Okamoto T. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). cells were two in and into of The cell using a in a and to using an The to by the addition of of in and the of an of of both in containing and for in an were and by or to with were to by an were using the of cells with and in 1 and for were by and by with protein for were to containing or to protein were 3 with and to with an One of a cell with or with containing a caveolin 1 peptide The were with and with and to with an were to by The and of GST fusion proteins as we previously P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). of the cytoplasmic of the EGF-R were by using containing and into the fusion proteins were purified by 1993; PubMed Scopus Google Scholar). proteins were by and to were with to protein and to with We to The were as previously J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of peptide in per well for the for or for were for 1 with containing fusion proteins S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google or in were to the of the cells were with Binding of GST fusion proteins using an by a Binding of using The by the addition of a the caveolin 1 a of the GST fusion protein and of peptide were The addition to the The that as We also in and binding using of purified EGF-R kinase with caveolin of and kinase were in a of of kinase (20 caveolin or EGF-R were in the the for 1 The by the addition of of for the by the addition of and for proteins were by or EGF-R cells, and kinase EGF-R cells were as for cells in the 1 and present the GST and 1 were to and with and of of this with of the were with and with containing The by using kinase activity using as the as suggested by the kinase were using of and 3 of in kinase (20 1 with a peptide the by the addition of of the by the addition of and for proteins were by or cells are known to both EGF-R A. J. T. A. A. J. T. J. J. E. M. P. PubMed Scopus Google and caveolin 1 (5Dupree P. Parton R.G. Raposo G. Kurzchalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar, G. I. S. J. Cell Biol. Google Scholar). Thus, we this cell to the of EGF-R with for the of membranes. that EGF-R with caveolin 1 in cells known as a to the EGF-R, also with caveolin In and proteins were these membrane has been in the formation of plasma is a cytosolic binding Caveolin 1 has been shown to interact with a variety of cytoplasmic signal molecules subunits, and a membrane-proximal region of NH2-terminal cytoplasmic domain. This 20-amino acid region 82–101) has been termed the caveolin scaffolding domain. it is not known caveolin with signaling as the interaction of EGF-R with caveolin we two independent (i) and (ii) the caveolin 1 scaffolding domain to that EGF-R with caveolin 1 a or caveolin 1 is EGF-R to were is in muscle and is not in cells, this as an negative for the role of the caveolin 1 scaffolding domain in this we cell with the caveolin 1 scaffolding domain to that this caveolin 1 peptide the EGF-R, binding of the caveolin 1 scaffolding domain in this Using the caveolin 1 scaffolding domain as a receptor to peptide two caveolin binding motifs were and is an or J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). two motifs are present in a in most G-protein α subunits, a known of caveolin-interacting proteins J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, we the cytoplasmic of the EGF-R and other receptor tyrosine kinases for a caveolin binding that a caveolin binding motif within the kinase domain of EGF-R and this motif is conserved within the kinase domains of most known receptor tyrosine kinases growth factor receptor, growth factor receptor and that this EGF-R is to the within a of the this EGF-R can as a for the caveolin scaffolding we fusion proteins of the cytoplasmic region of EGF-R 3 In addition, we also a peptide this EGF-R-derived caveolin binding motif (termed residues of that this caveolin binding is within and fusion proteins but is the fusion Using an that we previously to the caveolin binding region of G-protein α subunits J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), we the of these fusion proteins to interact with the caveolin 1 scaffolding domain. this we caveolin the scaffolding domains of caveolin 1 or caveolin or an caveolin 1 peptide in the fusion proteins with the caveolin 1 scaffolding domain but not with the caveolin scaffolding domain or with the caveolin 1 In addition, fusion not show an interaction with of these these results indicate the caveolin binding region of EGF-R between EGF-R residues and of the binding of and mapping are as the of a caveolin binding motif between EGF-R residues this EGF-R region in caveolin we the binding of a of the peptide to the caveolin 1 scaffolding domain. that the peptide with the caveolin 1 scaffolding a peptide within the receptor also a interaction with the caveolin 1 scaffolding domain. It has been previously that residues within the caveolin binding motif are for interaction with caveolin J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In with these a EGF-R peptide in residues were to to interact with the caveolin 1 scaffolding domain we the of these EGF-R and to with the EGF-R kinase domain for binding to the caveolin 1 scaffolding domain. In this the EGF-R and were in of the fusion protein to inhibition of the interaction of the kinase domain with the caveolin 1 scaffolding domain. that and were to for binding to caveolin the residues G-protein α subunits represent a well defined of caveolin-interacting proteins. that the EGF-R kinase domain with caveolin 1 in a as for Thus, we the of these receptor tyrosine kinase and to for G-protein binding to the caveolin 1 scaffolding domain. peptide the caveolin binding domain of as a and were as as a peptide in for the binding of a protein This the of residues within the EGF-R as two EGF-R residues to with the fusion protein the of the interaction of caveolin 1 with EGF-R, we the of the caveolin 1 scaffolding domain the autophosphorylation of a purified form of the EGF-R kinasein vitro. that the caveolin 1 scaffolding domain the kinase activity of inhibition a peptide of 3 and inhibition the caveolin 1 scaffolding domain inhibit the EGF-R One is that this caveolin-mediated inhibition is to tyrosine of caveolin or caveolin-derived in a form of inhibition of the EGF-R kinase. In of this the caveolin 1 scaffolding domain residues 82–101) contains both activity and two tyrosine the for tyrosine in this we a caveolin 1 scaffolding domain in both of these were to termed that the caveolin 1 scaffolding domain tyrosine as as the peptide in the autophosphorylation of In addition, the the in binding to the EGF-R peptide results that tyrosine of the caveolin 1 scaffolding domain 82–101) is not for activity of the EGF-R kinase by the caveolin 1 scaffolding domain could be by the direct interaction of caveolin with the kinase domain. In of this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of the 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 Caveolin 1 is the of a of caveolin Thus, we the activity of the scaffolding domains of caveolins and 3 in that the scaffolding domains of caveolins 1 and 3 both inhibit the EGF-R kinase the caveolin scaffolding domain has or This caveolin-mediated inhibition to be to a direct interact with the EGF-R kinase as the caveolin scaffolding domain also to interact with the region of the EGF-R kinase domain in the scaffolding domains of caveolins 1 and 3 both a results that the scaffolding domain of caveolin has protein for interaction with other molecules and the of these also of the caveolin 1 scaffolding domain for binding to the within the acid caveolin 1 scaffolding domain a with and for interaction with the fusion protein (termed or the EGF-R are in to the caveolin 1 the of is Two approaches were to residues within the caveolin 1 scaffolding domain that are for the interaction with the EGF-R kinase domain. First, of the region that a of amino is for inhibition of EGF-R kinase activity a of amino is for binding results are with the that binding to the kinase domain is to mediate inhibition of the EGF-R kinase, but that binding is not sufficient to mediate In addition, these are the that these two and kinase can be using the acid caveolin 1 scaffolding domain. that a of amino is for interaction of the caveolin 1 scaffolding domain with both the peptide and with the region of EGF-R domain the as in caveolin and in caveolin this may explain the EGF-R kinase domain is by the scaffolding domains of caveolins 1 and 3 but not by caveolin 2. Importantly, this region has also been shown to be for the interaction of caveolin 1 with of caveolin-interacting proteins, namely G-protein α that in cells the EGF-R and caveolin 1 this represent the activated or inactive of the this we an that recognizes the activated form of activated EGF-R is also to the containing results that activated EGF-R may also have the to interact with this we kinase activity is for EGF-R to interact with this we cells with EGF-R or with a kinase form of cells as a negative these cell lines were and as the for binding to the caveolin 1 scaffolding domain by a 1 fusion that both and kinase EGF-R interact with the GST fusion protein the caveolin scaffolding domain but not with GST binding of EGF-R to caveolin also with cells, that both inactive and activated of EGF-R have the to interact with caveolin results that the interaction is independent of receptor tyrosine autophosphorylation and receptor kinase In of our and R.G.W. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (403) Google have shown that EGF-R within caveolae for to in In this we have that the caveolin scaffolding domain can inhibit the autophosphorylation of a receptor tyrosine kinase, we have shown that caveolin binding both in vitro and in that activity of tyrosine kinases as c-Src and S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). it caveolin can interact with serine/threonine kinases. this we the of the caveolin scaffolding domain protein kinase C, a well serine/threonine kinase. We (i) most of the a caveolin binding motif within their kinase domain that is to the caveolin binding motif found in tyrosine kinases residues in and (ii) have been previously to caveolae by cell and of cell plasma R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar, R.G.W. J. Cell Biol. 1995; PubMed Scopus Google Scholar). these we the of purified termed this of and is by of with not or for kinase activity using as the that a peptide the caveolin 1 scaffolding domain the serine/threonine kinase activity of purified This inhibition is of with an of this is to the activity of other well of PubMed Scopus Google Scholar). for the autophosphorylation of EGF-R, the scaffolding domain of caveolin the caveolin 3 scaffolding domain is a inhibitor. In addition, it that the 20-amino acid caveolin 1 scaffolding domain 82–101) is for inhibition of as the peptide of the caveolin scaffolding domain can inhibit both tyrosine and serine/threonine kinases with this caveolin-derived protein module may represent a of general kinase inhibitor. In of this a caveolin binding motif is present within a conserved region of the kinase domain of most known protein kinases J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This region has been previously been termed region and is of protein kinase domain of major conserved that have been previously defined and are by T. PubMed Scopus Google Scholar). kinases play an role in the of growth and Two general of tyrosine kinases have been and kinases T. PubMed Scopus Google Scholar, T. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). tyrosine kinases, as EGF-R, are activated by the binding of to the protein tyrosine kinases be activated Both of tyrosine kinases can function as mutationally activated T. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, J. A. Full Text PDF PubMed Scopus Google Scholar). In this we report the direct interaction of the EGF-R with EGF-R is the receptor to be shown to interact with the of the interaction are to the interaction of caveolin with other caveolin-interacting proteins as G-protein α This that caveolin may interact with of signaling molecules a with G-protein α subunits, the caveolin binding domain within EGF-R the of residues to the scaffolding domain of caveolin the caveolin binding region of G-protein α subunits is located the of the the caveolin binding of EGF-R is located within a conserved region of the tyrosine kinase domain. the caveolin scaffolding domain inhibit the activity of the EGF-R One is that caveolin binding the receptor kinase in an inactive caveolin binding could receptor and as a of caveolin also the activity of other signaling molecules that not for activation as G-protein α we the Several independent lines of evidence are with a direct interaction between a receptor tyrosine kinase, EGF-R, and (i) tyrosine kinases and interact with and the kinase domains of tyrosine kinases and receptor tyrosine kinase are S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, 1997; PubMed Scopus Google Scholar, F. I. J. 1997; PubMed Scopus Google Scholar). (ii) receptor tyrosine kinases and growth factor receptor have now been shown to with caveolae or caveolae-like membrane domains P. R.G.W. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, R.G.W. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (403) Google Scholar, S. R.G.W. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar, J. Cell. PubMed Scopus Google Scholar), suggesting that a structural that is to this protein protein kinase domain of major conserved that have been previously defined and are by T. PubMed Scopus Google Scholar). caveolin binding motif is present within conserved region the function of this conserved region caveolin functions as a general kinase of this conserved caveolin binding In of this we show that the caveolin scaffolding domain can inhibit both tyrosine and serine/threonine kinases with Thus, this caveolin-derived protein module may represent a of general kinase inhibitor. Interaction of signaling molecules with caveolin or their could explain the cross-talk between receptor tyrosine kinases and receptor the role of EGF-R in signaling by the of the receptor has been in A. 1996; PubMed Scopus Google Scholar). the EGF-R signaling is located in these cells in caveolae R.G.W. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (403) Google Scholar), and we have previously shown that and receptor are with caveolin in cells M. U. Lisanti M.P. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). binding to is known to be sufficient to tyrosine kinases I. G. S. S. J. 1996; PubMed Scopus Google Scholar). In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network. We and Li for and and for cell lines and the