Immunoisolation of Caveolae with High Affinity Antibody Binding to the Oligomeric Caveolin Cage
TOWARD UNDERSTANDING THE BASIS OF PURIFICATION*
- From the Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 and the Sidney Kimmel Cancer Center, San Diego, California 92121
Abstract
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.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants HL52766 and HL58216 and a Grant-in-Aid from the American Heart Association. This work was presented at the 1997 ASCB meeting (Oh, P., and Schnitzer, J. E. (1997) Mol. Biol. Cell 8, 207a).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Supported by an Established Investigator award from the American Heart Association and Genentech. To whom correspondence should be addressed: Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. Tel.: 619-450-5990; Fax: 619-450-3251; E-mail: jschnitzer@skcc.org.
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↵2 P. Oh and J. E. Schnitzer, unpublished observations.
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↵3 D. McIntosh and J. E. Schnitzer, unpublished observations.
- Abbreviations:
- GPI
-
glycosylphosphatidylinositol
- TRM
-
Triton-resistant membranes
- VAMP
-
vesicle associated membrane protein (synaptobrevin)
- PAGE
-
polyacrylamide gel electrophoresis
- BSA
-
bovine serum albumin
- P
-
the silica-coated luminal endothelial cell plasma membranes isolated from rat lungs
- H
-
rat lung homogenate
- V
-
caveolar fraction isolated by shearing of silica-coated endothelial cell plasma membranes (P)
- P-V
-
repelleted silica-coated membranes stripped of caveolae by shearing
- PC
-
low density caveolae-enriched fraction isolated after sonication of silica-coated endothelial cell plasma membranes (P)
- P-C
-
repelleted silica-coated membranes stripped of caveolae by sonication
- PBS
-
phosphate-buffered saline
- SM
-
aliquot of starting material (usually V, PC, or AC) before immunoisolation
- UB
-
unbound fraction containing material not binding to immunoaffinity beads
- BD
-
bound fraction with material binding to immunoaffinity beads
- PM
-
plasma membrane fraction derived from rat lung using Percoll density centrifugation
- MBS
-
Mes-buffered saline
- Mes
-
2-(N-morpholino)ethanesulfonic acid
- CAV
-
caveolin monoclonal antibody (clone 2234)
- eNOS
-
endothelial nitric oxide synthase
- uPAR
-
urokinase plasminogen activator receptor
- ACE
-
angiotensin-converting enzyme
- 5′NT
-
5′-nucleotidase
- AC
-
low density caveolar fraction isolated after sonication of PM
- HD
-
high density membranes left after sonication of PM
- OC
-
caveolae immunoisolated using simplified procedure
- pAb
-
polyclonal antibody
- ELISA
-
enzyme-linked immunosorbent assay
- Tricine
-
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
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- Received August 31, 1998.
- Revision received May 10, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
