Melusin Is a New Muscle-specific Interactor for β1Integrin Cytoplasmic Domain

doi:10.1074/jbc.274.41.29282

Melusin Is a New Muscle-specific Interactor for β₁Integrin Cytoplasmic Domain*

From the ^‡Department of Genetics, Biology and Biochemistry, University of Torino, Torino 10126, Italy, the ^¶Immunogenetic and Experimental Oncology Center, CNR, Torino 10126, Italy, and the ^‖Institute of Genetics, University of Bari, Bari 70122, Italy

Abstract

Here we describe the isolation and partial characterization of a new muscle-specific protein (Melusin) which interacts with the integrin cytoplasmic domain. The cDNA encoding Melusin was isolated in a two-hybrid screening of a rat neonatal heart library using β₁A and β₁D integrin cytoplasmic regions as baits. Melusin is a cysteine-rich cytoplasmic protein of 38 kDa, with a stretch of acidic amino acid residues at the extreme carboxyl-terminal end. In addition, putative binding sites for SH3 and SH2 domains are present in the amino-terminal half of the molecule. Chromosomic analysis showed that melusin gene maps at Xq12.1/13 in man and in the synthenic region X band D in mouse. Melusin is expressed in skeletal and cardiac muscles but not in smooth muscles or other tissues. Immunofluorescence analysis showed that Melusin is present in a costamere-like pattern consisting of two rows flanking α-actinin at Z line. Its expression is up-regulated duringin vitro differentiation of the C2C12 murine myogenic cell line, and it is regulated during in vivo skeletal muscle development. A fragment corresponding to the tail region of Melusin interacted strongly and specifically with β₁ integrin cytoplasmic domain in a two-hybrid test, but the full-length protein did not. Because the tail region of Melusin contains an acidic amino acid stretch resembling high capacity and low affinity calcium binding domains, we tested the possibility that Ca²⁺ regulates Melusin-integrin association. In vitro binding experiments demonstrated that interaction of full-length Melusin with detergent-solubilized integrin heterodimers occurred only in absence of cations, suggesting that it can be regulated by intracellular signals affecting Ca²⁺ concentration.

Footnotes

↵* This work was supported by Grants 851 (to G. T.) and E.672 (to M. R.) from Theleton, from the Ministry of University and Scientific Research (to F. A.), from the National Research Council (P. F. Biotecnologie) (to F. A.) and from ASI (Italian Space Agency Grant 98-112).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) and .
↵§ To whom correspondence should be addressed: Dept. of Genetics, Biology and Biochemistry, Via Santena 5 bis, 10126 Torino, Italy. Tel.: 39-011-6706680; Fax: 39-011-6706547; E-mail: brancacc@molinette.unito.it.
Abbreviations:

PCR

polymerase chain reaction

dbEST

data base of expressed sequence tags

GST

glutathione S-transferase

MBP

maltose binding protein

bFGF

bovine fibroblast growth factor

nt

nucleotide(s)

DAPI

4,6-diamidino-2-phenylindole

PBS

phosphate-buffered saline

TBS

Tris-buffered saline

TIU

trypsin inhibitory units

BSA

bovine serum albumin

Cterm

carboxyl-terminal
- Received April 22, 1999.
- Revision received July 12, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.