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 alpha subunits.
We endeavored to identify proteins interacting with KLGFFKR, a highly conserved motif in the cytoplasmic domain adjacent to the transmembrane domain of the alpha 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 alpha 3 and alpha 5 subunits but not with the beta 1 subunit. Furthermore, p60 could be copurified with alpha 3 beta 1 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 alpha subunits. Our data suggest that p60 (Ro/SS-A Ag) can specifically bind to integrin alpha subunits via the highly conserved KLGFFKR amino acid sequence.
Rojiani
M V
MV
Department of Advanced Therapeutics, British Columbia Cancer Agency, Vancouver, Canada.
Finlay
B B
BB
Gray
V
V
Dedhar
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D037282:Calreticulin; D002460:Cell Line; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000737:chemistry*; D006801:Humans; D016023:Integrins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009447:Neuroblastoma / Q000737:chemistry; D012516:Osteosarcoma / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D020733:RNA, Small Cytoplasmic; D011817:Rabbits; D012261:Ribonucleoproteins; D012689:Sequence Homology, Nucleic Acid; D000099207:SS-A Antigen
D001324:Autoantigens; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D016023:Integrins; D010455:Peptides; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human