IDENTIFICATION OF THE CLEAVAGE SITES IN THE ALPHA-6A INTEGRIN SUBUNIT- STRUCTURAL REQUIREMENTS FOR CLEAVAGE AND FUNCTIONAL-ANALYSIS OF THEUNCLEAVED ALPHA-6A-BETA-1 INTEGRIN

The alpha 6A and alpha 6B integrin subunits are proteolytically cleave d 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 stru cture 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 d ifferentially glycosylated products of one polypeptide. Rather they po ssess different polypeptide backbones, which presumably result from pr oteolytic cleavage at distinct sites in the alpha 6 precursor. Mutatio ns were introduced in the codons for the (RKKR879)-K-876, (EK884)-K-88 3, (RK891)-K-890 and (RK899)-K-898 sequences, the potential proteolyti c cleavage sites, and wild-type and mutant alpha 6A cDNAs were transfe cted into K562 cells. The mutant alpha 6A integrin subunits were expre ssed in association with endogenous beta 1 at levels comparable to tha t of wild-type alpha 6A beta 1. A single alpha 6 polypeptide chain (15 0 kDa) was precipitated from transfectants expressing alpha 6A with mu tations or deletions in the RKKR sequence. Mutations in the EK sequenc e 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 tw o RK sequences were, like wild-type alpha 6A, cleaved into one heavy a nd two light chains. Thus a change in the RKKR sequence prevents the c leavage of alpha 6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing o f the N-termini of the two alpha 6A light chains from platelets demons trated 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 ess ential for cleavage of RKKR. alpha 6A mutants with the RKKR sequence s hifted 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 s hifted to other positions in the alpha 6A subunit, including one in wh ich it was shifted two residues farther than the EK cleavage site, wer e not cleaved. In addition, alpha 6A mutants with an alpha 5-like clea vage 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 al pha 6A beta 1 integrins bound to laminin-1. The phorbol ester PMA, whi ch activates cleaved wild-type and mutant alpha 6A beta 1, did not act ivate uncleaved alpha 6A beta 1. Thus uncleaved alpha 6A beta 1 is cap able of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of alpha 6 is required to generate a proper conf ormation that enables the affinity modulation of the alpha 6A beta 1 r eceptor by PMA.