T-cadherin Is a Major Glycophosphoinositol-anchored Protein Associated with Noncaveolar Detergent-insoluble Domains of the Cardiac Sarcolemma

doi:10.1074/jbc.273.12.6937

T-cadherin Is a Major Glycophosphoinositol-anchored Protein Associated with Noncaveolar Detergent-insoluble Domains of the Cardiac Sarcolemma*

From the ^‡Department of Medicine and ^§Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637 and ^¶Burnham Research Institute, La Jolla, California

Abstract

Sucrose-density flotation analysis of Triton-insoluble membrane domains isolated from highly purified sheep ventricular sarcolemma revealed the presence of two major 120- and 100-kDa proteins. Both species migrated in two-dimensional isoelectric focussing/SDS gels with an apparent pI of ∼4.3, suggesting that they might be related. Microsequence analysis of peptides derived from the 100-kDa protein yielded amino acid sequences with high homology to T-cadherin, a truncated cadherin lacking a cytoplasmic domain. The similarity was confirmed using antibodies to chicken T-cadherin that reacted with both proteins on immunoblots. T-cadherin was released from the detergent-insoluble sarcolemmal fraction by phospholipase C treatment indicating that it is linked to the membrane by a glycophosphoinositol anchor. T-cadherin could be ADP-ribosylated by a transferase that was also present in the caveolin-enriched Triton-insoluble fraction. T-cadherin-containing membrane fragments cofractionated on sucrose gradients with caveolin-3, a marker protein for myocyte caveolae. However, immunopurified caveolin-3-containing membranes contained no associated T-cadherin. Immunocytochemical analysis of cultured rat atrial myocytes revealed that T-cadherin and caveolin have related but nonoverlapping staining patterns. These results suggest that T-cadherin is a major glycophosphoinositol-linked protein in cardiac myocytes and that it may be located in plasma membrane “rafts” distinct from but possibly adjacent to caveolae.

Footnotes

↵* This work was supported by Grants from the American Heart Association (to H. C. P.) and from the National Institutes of Health (HL-10503 and HL-54302) (to E. P.).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.
↵‖ To whom correspondence should be addressed: Dept. of Pharmacological and Physiological Sciences, The University of Chicago, 947 E. 58th St., Chicago, IL 60637. Tel.: 773-702-9335; Fax: 773-702-5903; E-mail: hpalfrey{at}midway.uchicago.edu.
↵1 The abbreviations used are: GPI, glycophosphoinositol; PI·PLC, phosphatidylinositol-specific phospholipase C; M, crude microsomes; SL, sarcolemma; T-CAV, Triton-insoluble caveolin-rich membranes; TIFF, Triton-insoluble floatable fraction; MES, 4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focussing.
- Received August 25, 1997.
- Revision received December 1, 1997.
The American Society for Biochemistry and Molecular Biology, Inc.