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(Received for publication, July 1, 1996, and in revised form, August 26, 1996)
From the Caveolae were originally defined morphologically
as 50-100 nm noncoated vesicular organelles located at or near the
plasma membrane. Caveolin, a vesicular integral membrane protein of 21
kDa, is a principal protein component of caveolae membranes in
vivo. Caveolin interacts with itself to form high molecular mass
oligomers, suggesting that it might play a structural role in the
formation of caveolae membranes. However, it remains controversial
whether recombinant expression of caveolin is necessary or sufficient
to generate caveolae membranes in vivo. To directly address
this issue, we have taken a different experimental approach by
exploiting a heterologous expression system. Here, we have
recombinantly expressed mammalian caveolin in Sf21 insect cells using
baculovirus-based vectors. Two isoforms of caveolin have been
identified that differ at their extreme N terminus;
Volume 271, Number 45,
Issue of November 8, 1996
pp. 28647-28654
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A MODEL SYSTEM FOR THE BIOCHEMICAL AND MORPHOLOGICAL STUDY OF
CAVEOLAE BIOGENESIS
,
,
,
The Whitehead Institute for Biomedical
Research, Cambridge, Massachusetts 02142-1479 and ¶ Hiroshima
University School of Medicine, Department of Biochemistry, 1-2-3
Kasumi, Minami-ku, Hiroshima 734, Japan
-caveolin
contains residues 1-178, and 
-caveolin contains residues 32-178.
After recombinant expression in Sf21 insect cells, both - and
-caveolin formed SDS-resistant high molecular mass oligomers of the
same size as native caveolin. Morphologically, expression of either
caveolin isoform resulted in the intracellular accumulation of a
homogeneous population of caveolae-sized vesicles with a diameter
between 50 and 120 nm (80.3 ± 14.8 nm). This indicates that each
caveolin isoform can independently generate these structures and that
caveolin residues 1-31 are not required for this process. Using
caveolin as a marker protein and a detergent-free procedure to purify
caveolae from mammalian cells, we purified these recombinant
caveolin-induced vesicles from insect cells. These purified recombinant
vesicles: (i) have the same buoyant density as mammalian
caveolae; (ii) appear as ~50-100 nm membranous structures by
whole-mount electron microscopy; and (iii) contain ~95% of the
recombinantly expressed caveolin protein by Western blotting.
Immuno-labeling of these structures with anti-caveolin IgG confirmed
that they contain caveolin. Thus, ectopic overexpression of caveolin in
this heterologous system is sufficient to drive the formation of
caveolae-like vesicles. Further functional analysis demonstrated that
caveolin was capable of interacting with a known caveolin-interacting
protein, Ha-Ras, when coexpressed in insect cells by co-infection with
two recombinant baculoviruses. Taken together, our results demonstrate
that baculovirus-based expression of caveolin in insect cells provides
an attractive experimental system for studying the biogenesis of
caveolae.
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J. Couet, S. Li, T. Okamoto, T. Ikezu, and M. P. Lisanti Identification of Peptide and Protein Ligands for the Caveolin-scaffolding Domain. IMPLICATIONS FOR THE INTERACTION OF CAVEOLIN WITH CAVEOLAE-ASSOCIATED PROTEINS J. Biol. Chem., March 7, 1997; 272(10): 6525 - 6533. [Abstract] [Full Text] [PDF]
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K. S. Song, Z. Tang, S. Li, and M. P. Lisanti Mutational Analysis of the Properties of Caveolin-1. A NOVEL ROLE FOR THE C-TERMINAL DOMAIN IN MEDIATING HOMO-TYPIC CAVEOLIN-CAVEOLIN INTERACTIONS J. Biol. Chem., February 14, 1997; 272(7): 4398 - 4403. [Abstract] [Full Text] [PDF]
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Z. Tang, T. Okamoto, P. Boontrakulpoontawee, T. Katada, A. J. Otsuka, and M. P. Lisanti Identification, Sequence, and Expression of an Invertebrate Caveolin Gene Family from the Nematode Caenorhabditis elegans. IMPLICATIONS FOR THE MOLECULAR EVOLUTION OF MAMMALIAN CAVEOLIN GENES J. Biol. Chem., January 24, 1997; 272(4): 2437 - 2445. [Abstract] [Full Text] [PDF]
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A. Schlegel and M. P. Lisanti A Molecular Dissection of Caveolin-1 Membrane Attachment and Oligomerization. TWO SEPARATE REGIONS OF THE CAVEOLIN-1 C-TERMINAL DOMAIN MEDIATE MEMBRANE BINDING AND OLIGOMER/OLIGOMER INTERACTIONS IN VIVO J. Biol. Chem., July 7, 2000; 275(28): 21605 - 21617. [Abstract] [Full Text] [PDF]
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A. Schlegel, P. Arvan, and M. P. Lisanti Caveolin-1 Binding to Endoplasmic Reticulum Membranes and Entry into the Regulated Secretory Pathway Are Regulated by Serine Phosphorylation. PROTEIN SORTING AT THE LEVEL OF THE ENDOPLASMIC RETICULUM J. Biol. Chem., February 2, 2001; 276(6): 4398 - 4408. [Abstract] [Full Text] [PDF]
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V. O. Rybin, X. Xu, M. P. Lisanti, and S. F. Steinberg Differential Targeting of beta -Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae. A MECHANISM TO FUNCTIONALLY REGULATE THE cAMP SIGNALING PATHWAY J. Biol. Chem., December 22, 2000; 275(52): 41447 - 41457. [Abstract] [Full Text] [PDF]
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M. del Carmen de Marco, L. Kremer, J. P. Albar, J. A. Martinez-Menarguez, J. Ballesta, M. A. Garcia-Lopez, M. Marazuela, R. Puertollano, and M. A. Alonso BENE, a Novel Raft-associated Protein of the MAL Proteolipid Family, Interacts with Caveolin-1 in Human Endothelial-like ECV304 Cells J. Biol. Chem., June 15, 2001; 276(25): 23009 - 23017. [Abstract] [Full Text] [PDF]
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H. Lee, S. E. Woodman, J. A. Engelman, D. Volonte', F. Galbiati, H. L. Kaufman, D. M. Lublin, and M. P. Lisanti Palmitoylation of Caveolin-1 at a Single Site (Cys-156) Controls Its Coupling to the c-Src Tyrosine Kinase. TARGETING OF DUALLY ACYLATED MOLECULES (GPI-LINKED, TRANSMEMBRANE, OR CYTOPLASMIC) TO CAVEOLAE EFFECTIVELY UNCOUPLES c-Src AND CAVEOLIN-1 (TYR-14) J. Biol. Chem., September 7, 2001; 276(37): 35150 - 35158. [Abstract] [Full Text] [PDF]
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B. Razani, J. A. Engelman, X. B. Wang, W. Schubert, X. L. Zhang, C. B. Marks, F. Macaluso, R. G. Russell, M. Li, R. G. Pestell, et al. Caveolin-1 Null Mice Are Viable but Show Evidence of Hyperproliferative and Vascular Abnormalities J. Biol. Chem., October 5, 2001; 276(41): 38121 - 38138. [Abstract] [Full Text] [PDF]
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B. Razani, T. P. Combs, X. B. Wang, P. G. Frank, D. S. Park, R. G. Russell, M. Li, B. Tang, L. A. Jelicks, P. E. Scherer, et al. Caveolin-1-deficient Mice Are Lean, Resistant to Diet-induced Obesity, and Show Hypertriglyceridemia with Adipocyte Abnormalities J. Biol. Chem., March 1, 2002; 277(10): 8635 - 8647. [Abstract] [Full Text] [PDF]
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