Amino acid motifs required for isolated beta cytoplasmic domains to regulate 'in trans' beta 1 integrin conformation and function in cell attachment

The role of beta cytoplasmic domains in regulating beta 1 integrin conforma tion and function in cell attachment is not fully understood. In this study , we tested the ability of transiently expressed beta cytoplasmic domains c onnected to an extracellular reporter domain to regulate 'in trans' the con formation of endogenous beta 1 integrins, and compared these effects on cel l attachment. We found that chimeric receptors containing either the beta 1 , beta 3 or beta 5 cytoplasmic domains inhibited the expression of the conf ormationally dependent 9EG7 and 12G10 epitopes on endogenous beta 1 integri ns. In contrast, chimeric receptors containing the beta 4 or alpha 5 cytopl asmic domain, or a control receptor lacking a cytoplasmic domain, had no ef fect. This inhibition occurred in a dose-dependent manner that required hig h levels of expression of the chimeric receptor. These results suggest that beta 1 integrin conformation can be regulated by conserved cytosolic inter actions involving beta cytoplasmic domains. This is further supported by ou r findings that mutations within amino acid moths conserved among these bet a cytoplasmic domains, specifically the NXXY, NPXY and TST-like moths, redu ced the ability of these chimeric receptors to regulate beta 1 integrin con formation. Interestingly, the chimeric receptors inhibited cell attachment in a similar dose-dependent manner and required intact NXXY, NPXY, and TST- like motifs. The beta 1 chimera also inhibited the binding of soluble fibro nectin to endogenous beta 1 integrins. Thus, the concomitant inhibition in the expression of conformation-dependent integrin epitopes, cell attachment and ligand binding by the chimeras, suggests that the expression of the 9E G7 and 12G10 epitopes correlates with integrin function. However, Mn2+, whi ch is an extracellular activator of integrin function, increased 9EG7 expre ssion to basal levels in the presence of the pr chimera, but did not rescue cell attachment to the same extent. Thus, although the beta 1 integrin con formation recognized by mAb 9EG7 may be required for cell attachment, it is not sufficient, suggesting that the beta chimeras may be inhibiting both l igand binding and post-ligand binding events required for cell attachment. In addition, the inhibitory effects of the chimeric receptors on cell attac hment were not reversed by the addition of the pharmacological agents that inhibit intracellular signals previously shown to inhibit integrin function . This finding, together with the requirement for high levels of the chimer ic receptors and the fact that mutations in the same conserved motifs in he terodimeric pr integrins have been reported to regulate beta 1 integrin con formation and function in cell attachment, suggest that beta cytoplasmic do mains regulate these processes by interacting with cytosolic factors and th at the regulatory effect of the chimeras may be due to their ability to tit rate proteins from endogenous integrins.