Identification of the cleavage sites in the alpha 6A integrin subunit: Structural requirements for cleavage and functional analysis of the uncleaved alpha 6A beta 1 integrin

The alpha 6A and alpha 6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the alpha 6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of alpha 6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the alpha 6 precursor. Mutations were introduced in the codons for the (RKKR879)-K-876, (EK884)-K-883, (RK891)-K-890 and (RK899)-K-898 sequences, the potential proteolytic cleavage sites, and wild-type and mutant alpha 6A cDNAs were transfected into K562 cells. The mutant alpha 6A integrin subunits were expressed in association with endogenous beta 1 at levels comparable to that of wild-type alpha 6A beta 1. A single alpha 6 polypeptide chain (150 kDa) was precipitated from transfectants expressing alpha 6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded alpha 6A subunits that were cleaved once into a heavy and a light chain, whereas alpha 6A subunits with mutations in one of the two RK sequences were, like wild-type alpha 6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of alpha 6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two alpha 6A light chains from platelets demonstrated that cleavage occurs after Arg(879) and Lys(884). Because alpha 6(RKKG), alpha 6(GKKR) and alpha 6(RGGR) subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. alpha 6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys(884), were partly cleaved, whereas alpha 6A mutants with the RKKR sequence shifted to other positions in the alpha 6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, alpha 6A mutants with an alpha 5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-beta 1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved alpha 6A beta 1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant alpha 6A beta 1, did not activate uncleaved alpha 6A beta 1. Thus uncleaved alpha 6A beta 1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of alpha 6 is required to generate a proper conformation that enables the affinity modulation of the alpha 6A beta 1 receptor by PMA.