PROTEOLYTIC PROCESSING OF ENDOGENOUS AND RECOMBINANT BETA(4) INTEGRIN SUBUNIT

The alpha-6-beta-4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta-4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta-4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta-4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta-4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta-4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta-4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta-4 are proteolytically processed. To examine the processing of the beta-4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain Of beta-4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta-4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta-4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta-4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha-6-beta-4 integrin.