Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin alpha-subunit-binding protein.

We have demonstrated recently that calreticulin, an intracellular calcium-binding protein, can interact with the alpha-subunits of integrin receptors via the highly conserved KXGFFKR amino acid sequence present in the cytoplasmic domains of all integrin alpha-subunits (Rojiani et al. (1991) Biochemistry 30, 9859-9866). Here we demonstrate that calreticulin can be co-localized by immunofluorescence as well as co-purified with integrins, that recombinant calreticulin can also interact with integrins, and that the interaction occurs predominantly via the N-domain of calreticulin, to a much lesser extent with the C-domain, but not at all with the proline-rich P-domain. To demonstrate a physiological role for the interaction of calreticulin with integrins, calreticulin expression was downregulated by treating cells with antisense oligonucleotides designed to inhibit the initiation of translation of calreticulin. Antisense oligonucleotides, but not sense or non-sense oligonucleotides, inhibited attachment and spreading of cells cultured in the presence of fetal bovine serum, and also of cells plated on individual extracellular matrix substrates in the absence of serum. The antisense oligonucleotide inhibited cell proliferation of anchorage-dependent cells slightly, but there was no effect on cell viability. The effect on cell attachment was similar to that achieved by treating cells with an antisense oligonucleotide designed to inhibit translation of the integrin alpha 3 subunit, which resulted in the inhibition of cell attachment to alpha 3 beta 1-specific substrates. The effect of the antisense calreticulin oligonucleotide on cell attachment was demonstrated to be integrin-mediated since antisense calreticulin treatment of Jurkat cells abrogated the stimulation of collagen cell attachment achieved by attachment-stimulating signalling anti-alpha 2 (JBS2) and anti-beta 1 (21C8) antibodies. The oligonucleotides did not affect the rate of cell proliferation of these cells. These results demonstrate a fundamental role of calreticulin in cell-extracellular matrix interactions.