In Vitro Interaction of a Polypeptide Homologous to Human Ro/SS-A Antigen (Calreticulin) with a Highly Conserved Amino Acid Sequence in the Cytoplasmic Domain of Integrin α Subunits

Mumtaz V. Rojiani, Virginia Gray, Shoukat Dedhar, B. Brett Finlay

Research output: Contribution to journalArticle

182 Scopus citations

Abstract

We endeavored to identify proteins interacting with KLGFFKR, a highly conserved motif in the cytoplasmic domain adjacent to the transmembrane domain of the α subunit of integrins. We found that affinity chromatography of cell extracts with this peptide followed by elution with EDTA resulted in the isolation of a 60-kDa protein (p60). The N-terminal amino acid sequence of this 60-kDa polypeptide was found to be highly homologous to the Ro/SS-A antigen, a 60-kDa protein homologous to calreticulin and Aplysia “memory molecule”. The binding of p60 was found to be specific for the KLGFFKR sequence since this polypeptide did not bind to a peptide with a scrambled amino acid sequence (KLRFGFK), and it was also specifically eluted from the KLGFFKR affinity matrix ith soluble KLGFFKR peptide but not with the scrambled peptide. Solid phase in vitro binding assays demonstrated specific interaction of p60 with integrin α3 and α5 subunits but not with the β1 subunit. Furthermore, p60 could be copurified with α3β1 following coincubation in vitro. These interactions could be inhibited by KLGFFKR peptide and also by EDTA, indicating sequence-specific and divalent cation dependent binding. Despite the fact that calreticulin is thought to be localized in the endoplasmic reticulum, a pool of Ro/SS A antigen homologous 60-kDa polypeptide was found to be present in the soluble cytoplasm, indicating the feasibility of an interaction of p60 with the integrin α subunits. Our data suggest that p60 (Ro/SS-A Ag) can specifically bind to integrin α subunits via the highly conserved KLGFFKR amino acid sequence.

Original languageEnglish (US)
Pages (from-to)9859-9866
Number of pages8
JournalBiochemistry
Volume30
Issue number41
DOIs
StatePublished - Oct 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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