Critical cysteine residues for regulation of integrin alphaIIbbeta3 are clustered in the epidermal growth factor domains of the beta3 subunit.

Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin beta3 subunit are involved in the regulation of alphaIIbbeta3. In the present study, we studied systematically the role of disulphide bonds in beta3 on the ligand-binding function of alphaIIbbeta3 by mutating individual cysteine residues of beta3 to serine. We found that the disulphide bonds that are critical for alphaIIbbeta3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate alphaIIbbeta3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated alphaIIbbeta3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep alphaIIbbeta3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction.

The role of the CPNKEKEC sequence in the beta(2) subunit I domain in regulation of integrin alpha(L)beta(2) (LFA-1).

The alpha(L) I (inserted or interactive) domain of integrin alpha(L)beta(2) undergoes conformational changes upon activation. Recent studies show that the isolated, activated alpha(L) I domain is sufficient for strong ligand binding, suggesting the beta(2) subunit to be only indirectly involved. It has been unclear whether the activity of the alpha(L) I domain is regulated by the beta(2) subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the beta(2) I domain with a corresponding beta(3) sequence, or mutating Lys(174) to Thr, constitutively activates alpha(L)beta(2) binding to ICAM-1. These mutants do not require Mn(2+) for ICAM-1 binding and are insensitive to the inhibitory effect of Ca(2+). We have also localized a component of the mAb 24 epitope (a reporter of beta(2) integrin activation) in the CPNKEKEC sequence. Glu(173) and Glu(175) of the beta(2) I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon beta(2) integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the alpha(L) I domain did not eliminate the mAb 24 epitope. This confirms that the alpha(L) I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the beta(2) subunit I domain. These results suggest that the CPNKEKEC sequence of the beta(2) I domain is involved in regulating the alpha(L) I domain.