Expression of Caveolin-3 in Skeletal, Cardiac, and Smooth Muscle Cells

doi:10.1074/jbc.271.25.15160

Expression of Caveolin-3 in Skeletal, Cardiac, and Smooth Muscle Cells

CAVEOLIN-3 IS A COMPONENT OF THE SARCOLEMMA AND CO-FRACTIONATES WITH DYSTROPHIN AND DYSTROPHIN-ASSOCIATED GLYCOPROTEINS*

From the ^‡ Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479,
^¶ Shriners Hospitals for Crippled Children, Massachusetts General Hospital, Department of Anesthesia, Harvard Medical School, Boston, Massachusetts 02114, and
^‴ The Mount Sinai School of Medicine, Department of Pathology, New York, New York 10029

^∥ To whom correspondence should be addressed:
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142-1479.
Tel.: 617-258-5225; Fax: 617-258-9872; E-mail: lisanti{at}wi.mit.edu

Abstract

Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21–24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 skeletal myoblasts in culture. Using differentiated C2C12 skeletal myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, α-sarcoglycan, and β-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.

Footnotes

↵^§ Funded by a Swiss National Science Foundation Fellowship.
↵^∥ Recipient of Fellowships from the Byotai-Taisha Foundation and the Mochida Memorial Foundation.
↵^” Recipient of National Institutes of Health Postdoctoral Fellowship CA-71326.
↵* This work was supported in part by National Institutes of Health FIRST Award GM-50443 (to M. P. L.), a grant from the Elsa U. Pardee Foundation (to M. P. L.), and a grant from the W. M. Keck Foundation to the Whitehead Fellows program (to M. P. L.). 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.
↵¹ The abbreviations used are:

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

Mes

4-morpholineethanesulfonic acid.
- Received February 28, 1996.
- Revision received April 5, 1996.