Calmodulin Binds to Specific Sequences in the Cytoplasmic Domain of C-CAM and Down-regulates C-CAM Self-association

doi:10.1074/jbc.271.3.1393

Calmodulin Binds to Specific Sequences in the Cytoplasmic Domain of C-CAM and Down-regulates C-CAM Self-association (*)

From the (1)Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-171 77 Stockholm, Sweden and
(2)Pharmacia Biotechnology AB, S-751 82 Uppsala, Sweden

^§ To whom correspondence should be addressed. Tel.: 46-8-728-73-00; Fax: 46-8-30-18-33; bjorn.obrink{at}cmb.ki.seb.ki.se.

Abstract

C-CAM is a cell adhesion molecule belonging to the immunoglobulin supergene family and is known to mediate calcium-independent homophilic cell-cell binding. Two major isoforms, C-CAM1 and C-CAM2, which differ in their cytoplasmic domains, have been identified. Previous investigations have demonstrated that both cytoplasmic domains can bind calmodulin in a calcium-dependent reaction. In this investigation, peptides corresponding to the cytoplasmic domains of C-CAM were synthesized on cellulose membranes and used to map the binding sites for I-labeled calmodulin. Both C-CAM1 and C-CAM2 had one strong calmodulin-binding site in the membrane-proximal region. These binding regions were conserved in C-CAM from rat, mouse, and man. In addition, C-CAM1 from rat and mouse contained a weaker binding site in the distal region of the cytoplasmic domain. Biosensor experiments were performed to determine rate and equilibrium constants of the C-CAM/calmodulin interaction. An association rate constant of 3.3 × 10⁵M s and two dissociation rate constants of 2.2 × 10 and 3.1 × 10 s were determined. These correspond to equilibrium dissociation constants of 6.7 × 10 and 9.4 × 10M, respectively. In dot-blot binding experiments, it was found that binding of calmodulin causes a down-regulation of the homophilic self-association of C-CAM. This suggests that calmodulin can regulate the functional activity of C-CAM.

Footnotes

↵* This work was supported by grants from the Swedish Medical Research Council (Project No. 05200), the Swedish Society for Medical Research, the Petrus and 8a Hedlunds Foundation, and the Karolinska Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CAMs

cell adhesion molecules

Fmoc

N-(9-fluorenyl)methoxycarbonyl

MOPS

4-morpholinepropanesulfonic acid

dansyl

5-dimethylaminonaphthalene-1-sulfonyl.
↵²I. Hunter and B. öbrink, unpublished observations.
- Received June 5, 1995.
- Revision received October 20, 1995.