Proteolytic processing of endogenous and recombinant β4 integrin subunit

Abstract

The α6β4 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 β4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the β4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of β4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave β4 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 β4 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 β4 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 β4 are proteolytically processed. To examine the processing of the β4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of β4. The distinct staining patterns obtained with the two types of antibodies provided evidence that β4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that β4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the β4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the α6β4 integrin.