Immunoisolation of Caveolae with High Affinity Antibody Binding to the Oligomeric Caveolin Cage
1999; Elsevier BV; Volume: 274; Issue: 33 Linguagem: Inglês
10.1074/jbc.274.33.23144
ISSN1083-351X
Autores Tópico(s)Ion Transport and Channel Regulation
ResumoDefining the molecular composition of caveolae is essential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-1α and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homogenates without using the silica-coating technology and (ii) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechanical disruption. Sonication of plasma membranes, isolated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolations to purify caveolae. Shearing of silica-coated plasma membranes provides a homogeneous population of caveolae whose constituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphosphatidylinositol-anchored proteins, β-actin, and angiotensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-1α. Caveolin-1β and -2 can also exist in these same caveolae. The isolation and analytical procedures as well as the time-dependent dissociation of signaling molecules from caveolae contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent preparation for use in the molecular analysis of caveolae. Defining the molecular composition of caveolae is essential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-1α and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homogenates without using the silica-coating technology and (ii) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechanical disruption. Sonication of plasma membranes, isolated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolations to purify caveolae. Shearing of silica-coated plasma membranes provides a homogeneous population of caveolae whose constituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphosphatidylinositol-anchored proteins, β-actin, and angiotensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-1α. Caveolin-1β and -2 can also exist in these same caveolae. The isolation and analytical procedures as well as the time-dependent dissociation of signaling molecules from caveolae contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent preparation for use in the molecular analysis of caveolae. glycosylphosphatidylinositol Triton-resistant membranes vesicle associated membrane protein (synaptobrevin) polyacrylamide gel electrophoresis bovine serum albumin the silica-coated luminal endothelial cell plasma membranes isolated from rat lungs rat lung homogenate caveolar fraction isolated by shearing of silica-coated endothelial cell plasma membranes (P) repelleted silica-coated membranes stripped of caveolae by shearing low density caveolae-enriched fraction isolated after sonication of silica-coated endothelial cell plasma membranes (P) repelleted silica-coated membranes stripped of caveolae by sonication phosphate-buffered saline aliquot of starting material (usually V, PC, or AC) before immunoisolation unbound fraction containing material not binding to immunoaffinity beads bound fraction with material binding to immunoaffinity beads plasma membrane fraction derived from rat lung using Percoll density centrifugation Mes-buffered saline 2-(N -morpholino)ethanesulfonic acid caveolin monoclonal antibody (clone 2234) endothelial nitric oxide synthase urokinase plasminogen activator receptor angiotensin-converting enzyme 5′-nucleotidase low density caveolar fraction isolated after sonication of PM high density membranes left after sonication of PM caveolae immunoisolated using simplified procedure polyclonal antibody enzyme-linked immunosorbent assay N -[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine In the last few years, there has been a burst of new research on defining the molecular architecture and function of one type of non-clathrin-coated plasmalemmal in various degrees on the surface of many mammalian cell types. In large measure, this surge of experimentation has resulted from the development of a number of techniques purporting to purify these invaginated microdomains from various cells and/or tissues. Cumulatively, the similarities between the lists of molecules identified in these preparations has been rather striking. It is interesting that this remarkable overall agreement has been minimally appreciated or acknowledged but rather only the few, yet significant differences have come to the forefront of discussion about this field. As with most fields growing rapidly in the modern era of research, this field has experienced its growing pains and controversies. Time resolves most scientific disputes, and here we wish to contribute new constructive information that may provide a firm basis for better understanding the basics of caveolae purification as well as the basis of both the similarities and differences found in the major methods currently devised for caveolae isolation. Defining the molecular composition of caveolae is a critical step in dissecting and ultimately defining their function. Two groups (1Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z. Hermanowski-Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (809) Google Scholar, 2Chang W.J. Ying Y.S. Rothberg K.G. Hooper N.M. Turner A.J. Gambliel H.A. De Gunzburg J. Mumby S.M. Gilman A.G. Anderson R.G. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (309) Google Scholar) reported purification of caveolae in 1994 using a methodology that evolved from the identification of caveolin (also called VIP21) as a marker protein of caveolae (3Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1859) Google Scholar, 4Kurzchalia T.V. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (462) Google Scholar, 5Dupree P. Parton R.G. Raposo G. Kurzchalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (400) Google Scholar) and the development of a subfractionation procedure for following the synthesis and trafficking of glycosylphosphatidylinositol (GPI)1-anchored proteins through the Golgi compartment (6Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2602) Google Scholar). Cells or tissue are homogenized and exposed to Triton X-100 at 4 °C before using centrifugation to isolate the low buoyant density Triton-resistant membranes (TRM) enriched in caveolin, GPI-anchored proteins, cytoskeletal proteins, and signaling molecules (1Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z. Hermanowski-Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (809) Google Scholar, 2Chang W.J. Ying Y.S. Rothberg K.G. Hooper N.M. Turner A.J. Gambliel H.A. De Gunzburg J. Mumby S.M. Gilman A.G. Anderson R.G. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (309) Google Scholar, 7Sargiacomo M. Sudol M. Tang Z.L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (858) Google Scholar). These findings contributed to the hypothesis that caveolae may be signaling organelles (8Lisanti M.P. Scherer P.E. Tang Z. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (588) Google Scholar) and that certain lipid modifications of proteins may target key signaling molecules to caveolae (9Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (340) Google Scholar, 10Robbins S.M. Quintrell N.A. Bishop J.M. Mol. Cell. Biol. 1995; 15: 3507-3515Crossref PubMed Scopus (227) Google Scholar, 11Garcia-Cardena G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (575) Google Scholar, 12Shaul P. Smart E. Robinson L. German Z. Yuhanna I. Ying Y. Anderson R. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Although a significant step forward, this methodology actually isolates caveolae along with other cell surface and intracellular microdomains, all with similar buoyant densities and detergent resistance (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar). Many vesicles in TRM are much larger than caveolae and are quite rich in GPI-anchored proteins but not caveolin (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar). Similar large Triton-resistant vesicles, which have the same buoyant density and are rich in GPI-anchored proteins, are readily isolated from cells apparently neither expressing caveolin nor having morphologically distinguishable caveolae (14Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar, 15Gorodinsky A. Harris D.A. J. Cell Biol. 1995; 129: 619-627Crossref PubMed Scopus (296) Google Scholar). Finally, although TRM contain most of the caveolin in the cell and caveolin is an excellent marker for caveolae at the cell surface, caveolin can exist elsewhere including the trans-Golgi network (4Kurzchalia T.V. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (462) Google Scholar, 5Dupree P. Parton R.G. Raposo G. Kurzchalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (400) Google Scholar). In 1994, caveolae were isolated directly from plasma membranes that first had been isolated away from other potential contaminants, including Golgi membranes, by using an in situ silica-coating procedure (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 16Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Mol. Biol. Cell. 1994; 5: 75aGoogle Scholar, 17Schnitzer J.E. McIntosh D. Oh P. Mol. Biol. Cell. 1994; 5: 320aGoogle Scholar, 18Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (230) Google Scholar, 19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). In this procedure, caveolae are sheared away from the silica-coated rat lung endothelial cell plasma membranes before isolation by sucrose density centrifugation. This method yields an apparently homogeneous population of low density vesicles that morphologically are similar in size and shape to caveolae and biochemically are enriched in various caveolar markers (caveolin, inositol 1, 4, 5-trisphosphate-like receptor, dynamin, sialoglycolipid GM1, and calcium ATPase) while being markedly depleted in noncaveolar markers (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 18Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (230) Google Scholar, 19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 20Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (552) Google Scholar, 21Schnitzer J.E. Born G.V.R. Schwartz C.J. Vascular Endothelium: Physiology, Pathology and Therapeutic Opportunities. 3. Schattauer, Stuttgart, Germany1997: 77-95Google Scholar). This subfractionation allows (i) caveolae to be isolated with or without detergent and (ii) plasmalemmal membranes rich in GPI-anchored proteins but devoid of caveolin to be isolated separately from caveolae rich in caveolin but not GPI-anchored proteins (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 16Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Mol. Biol. Cell. 1994; 5: 75aGoogle Scholar, 17Schnitzer J.E. McIntosh D. Oh P. Mol. Biol. Cell. 1994; 5: 320aGoogle Scholar, 18Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (230) Google Scholar, 19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Some caveolar signaling molecules, especially those with lipid anchors, are solubilized to various degrees by Triton X-100 (22Liu J. Oh P. Horner T. Rogers R.A. Schnitzer J.E. J. Biol. Chem. 1997; 272: 7211-7222Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). To confirm that molecules thus found in caveolae are indeed in the same caveolae coated with caveolin, a process was developed in 1995 (19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) using magnetic beads to immunoisolate caveolae. Over the last 4 years, this immunoaffinity isolation has verified the existence of many different proteins in caveolae including signaling proteins activated by growth factors (22Liu J. Oh P. Horner T. Rogers R.A. Schnitzer J.E. J. Biol. Chem. 1997; 272: 7211-7222Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), the fusion protein synaptobrevin (also known as VAMP) (19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), and dynamin (20Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (552) Google Scholar). Immunoisolations have also been performed on transfected cells expressing recombinant tagged caveolins (23Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). As it became apparent that TRM are not purified caveolae, Anderson and colleagues (24Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar) developed an alternative procedure that first isolates a plasma membrane-rich fraction on a Percoll gradient and then sonicates these membranes to release caveolae for collection in low density fractions after OptiPrep-gradient centrifugation. This isolate was reported to be nearly identical in composition to TRM except for the avoidance of detergent extraction of a few signaling molecules such as G protein subunits (24Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar). It is quite enriched in caveolin, GPI-anchored proteins, and various signaling molecules. Sonication with sodium carbonate treatment has been used to isolate a low density caveolar fraction containing caveolin and signaling molecules but not GPI-anchored proteins (23Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). Finally, caveolae have been isolated under conditions that avoid physical detachment but rather utilize physiological release by adding GTP to induce caveolar budding from plasma membranes (25Schnitzer J.E. McIntosh D.P. Oh P. Science. 1996; 274 (Correction): 239-242Science. 1996; 274 (Correction)1069Crossref PubMed Scopus (212) Google Scholar). Dynamin at the neck of caveolae hydrolyzes GTP to release free caveolar transport vesicles that can be isolated by density centrifugation and are rich in caveolin and other caveolar markers but not GPI-anchored proteins (20Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (552) Google Scholar, 25Schnitzer J.E. McIntosh D.P. Oh P. Science. 1996; 274 (Correction): 239-242Science. 1996; 274 (Correction)1069Crossref PubMed Scopus (212) Google Scholar). From these methodologies, there has been general agreement from many laboratories that signaling molecules can be found in caveolae (1Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z. Hermanowski-Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (809) Google Scholar, 2Chang W.J. Ying Y.S. Rothberg K.G. Hooper N.M. Turner A.J. Gambliel H.A. De Gunzburg J. Mumby S.M. Gilman A.G. Anderson R.G. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (309) Google Scholar,7Sargiacomo M. Sudol M. Tang Z.L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (858) Google Scholar, 8Lisanti M.P. Scherer P.E. Tang Z. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 9Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (340) Google Scholar, 10Robbins S.M. Quintrell N.A. Bishop J.M. Mol. Cell. Biol. 1995; 15: 3507-3515Crossref PubMed Scopus (227) Google Scholar, 13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 15Gorodinsky A. Harris D.A. J. Cell Biol. 1995; 129: 619-627Crossref PubMed Scopus (296) Google Scholar, 19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 21Schnitzer J.E. Born G.V.R. Schwartz C.J. Vascular Endothelium: Physiology, Pathology and Therapeutic Opportunities. 3. Schattauer, Stuttgart, Germany1997: 77-95Google Scholar, 22Liu J. Oh P. Horner T. Rogers R.A. Schnitzer J.E. J. Biol. Chem. 1997; 272: 7211-7222Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 23Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 24Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar, 26Huang C. Hepler J.R. Chen L.T. Gilman A.G. Anderson R.G.W. Mumby S.M. Mol. Biol. Cell. 1997; 8: 2365-2378Crossref PubMed Scopus (188) Google Scholar, 27Liu P. Anderson R.G.W. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 28Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Moreover, various different assays show that caveolin can interact directly with various signaling molecules including G proteins and eNOS (29Li 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 (556) Google Scholar, 30Li S. Coute J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 31Garcia-Cardena G. Martasek P. Masters B.S.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 32Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 33Rizzo V. McIntosh D.P. Oh P. Schnitzer J.E. J. Biol. Chem. 1998; 273: 34724-34729Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Caveolin may act as a scaffolding protein that regulates signaling molecules by preferentially binding them in an inactive state (23Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 29Li 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 (556) Google Scholar, 30Li S. Coute J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 31Garcia-Cardena G. Martasek P. Masters B.S.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 32Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 33Rizzo V. McIntosh D.P. Oh P. Schnitzer J.E. J. Biol. Chem. 1998; 273: 34724-34729Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Despite these data, one report in 1997 (34Stan R.-V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (176) Google Scholar) concluded differently using a methodology that mixed different aspects of past procedures (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar,24Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar) in a new arrangement to immunoisolate caveolae. Instead of shearing, sonication was used in this study to dislodge caveolae from silica-coated rat lung endothelial cell plasma membranes before isolating the low density, caveolin-enriched vesicles by sucrose gradient centrifugation. This fraction was subjected to immunoisolation overnight using a rabbit polyclonal antiserum raised to a synthetic peptide similar to N-terminal region of caveolin. Although several signaling molecules were concentrated in low density, caveolin-rich vesicles released by sonication, little to no signal for many of these molecules such as eNOS and G proteins was reported in the immunoisolated caveolae. It was concluded from the data shown using volume equivalent analysis that caveolae are not signaling centers. In the end, these latter findings have extended uncertainty in the field from what had been a growing consensus about the lack of concentration of GPI-anchored proteins in caveolae under non-perturbed conditions to now the possible lack of many, if not all, lipid-anchored and possibly other signaling molecules in caveolae. This issue becomes of greater concern because many of the functions ascribed to caveolae, such as signaling, mechanotransduction (21Schnitzer J.E. Born G.V.R. Schwartz C.J. Vascular Endothelium: Physiology, Pathology and Therapeutic Opportunities. 3. Schattauer, Stuttgart, Germany1997: 77-95Google Scholar, 35Rizzo V. Sung A. Oh P. Schnitzer J.E. J. Biol. Chem. 1998; 273: 26323-26329Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), and even vesicular transport (19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 21Schnitzer J.E. Born G.V.R. Schwartz C.J. Vascular Endothelium: Physiology, Pathology and Therapeutic Opportunities. 3. Schattauer, Stuttgart, Germany1997: 77-95Google Scholar, 25Schnitzer J.E. McIntosh D.P. Oh P. Science. 1996; 274 (Correction): 239-242Science. 1996; 274 (Correction)1069Crossref PubMed Scopus (212) Google Scholar), may rely on the presence of key molecules discovered to be in caveolae through the use of these subfractionation techniques. In this work, we identify a high affinity, caveolin-specific monoclonal antibody that rapidly binds the oligomeric caveolin cage surrounding caveolae. By using this antibody, we have developed a method not only for rapidly purifying caveolae from cell and tissue homogenates but also for testing the purity of caveolae isolated using different methodologies. We identify factors that are important in purifying and analyzing caveolae and that explain the few but key compositional differences detected in various caveolar preparations. Antibodies against caveolin were purchased from Transduction Laboratories (Lexington, KY) (rabbit polyclonal (pAb) and mouse monoclonal (clone 2297 and 2234)) and from Zymed Laboratories Inc. (South San Francisco, CA) (mouse monoclonal (Z034)). The M-450 Dynabeads were purchased from Dynal (New Hyde Park, NY). All other reagents/supplies were obtained as in our past work (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar,18Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (230) Google Scholar, 19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 25Schnitzer J.E. McIntosh D.P. Oh P. Science. 1996; 274 (Correction): 239-242Science. 1996; 274 (Correction)1069Crossref PubMed Scopus (212) Google Scholar). As described previously (13Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar,18Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (230) Google Scholar, 19Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 25Schnitzer J.E. McIntosh D.P. Oh P. Science. 1996; 274 (Correction): 239-242Science. 1996; 274 (Correction)1069Crossref PubMed Scopus (212) Google Scholar), the proteins of various tissue fractions were solubilized and separated by SDS-PAGE for direct analysis by silver staining or for Western analysis by electrotransfer to nitrocellulose filters followed by immunoblotting using enhanced chemiluminescence and densitometric quantification using ImageQuant. Briefly, nitrocellulose filters from each gel were probed using primary antibody (diluted from 1:500 to 1:5000 in Blotto (5% nonfat dry milk in Tris-buffered saline with 0.5% Tween 20)) followed by the appropriate horseradish peroxidase-labeled reporter antibodies (diluted 1:1000). Protein concentrations were measured using the micro-bicinchoninic acid protein assay kit with bovine serum albumin (BSA) as a standard. The luminal endothelial cell plasma membranes and caveolae were isolated directly from rat lung tissue using an in situ silica-coating procedure as described in detail (36Oh P. Schnitzer J.E. Celis J. Cell Biology: A Laboratory Handbook. 2. Academic Press, Orlando1998: 34-46Google Scholar). Briefly, the rat lungs were perfused via the pulmonary artery with a colloidal silica solution to coat the luminal surface of the endothelium and allow selective isolation of the silica-coated endothelial cell plasma membranes (P) from the lung homogenate (H) by centrifugation. The caveolae are separated from P by shearing and then isolated by sucrose density centrifugation in a low buoyant density fraction (V) well separated from the silica-coated membrane pellet stripped of caveolae (P-V). Alternatively, as described in Ref. 34Stan R.-V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (176) Google Scholar, caveolae are separated from P by sonication and then isolated by sucrose density gradient centrifugation in a low buoyant density fraction (PC) away from the repelleted and sonication-stripped silica-coated membranes (P-C). The reactivity of caveolin antibodies with the silica-coated endothelial cell plasma membranes (P) was assessed by ELISA. Briefly, equal aliquots of P (5 μg in 100 μl) were placed in each well of a 96-well tray for drying overnight. After washing, the wells were blocked for 1 h with wash buffer (2% ovalbumin and 2 mm CaCl2 in phosphate-buffered saline (PBS)), incubated with wash buffer alone or containing antibody, washed, incubated with reporter antibody conjugated to horseradish peroxidase (1:500), and washed again. A substrate solution (50 mmNa2HPO4, 25 mm citric acid, 1.2 mg/ml o -phenylenediamine dihydrochloride, and 0.03% H2O2) was added. The
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