CLEAVAGE OF THE ALPHA-6A SUBUNIT IS ESSENTIAL FOR ACTIVATION OF THE ALPHA-6A-BETA-1 INTEGRIN BY PHORBOL 12-MYRISTATE 13-ACETATE

The alpha 6 integrin subunit is proteolytically cleaved during biosynt hesis in a covalently associated heavy and light chain, To examine the importance of cleavage for the function of the alpha 6 subunit, we in troduced mutations in the cDNA encoding the RKKR (876-879) sequence, t he presumed cleavage site, in which either one or two basic residues w ere replaced by glycine. Wild-type and mutant alpha 6A cDNAs (alpha 6( GKKR), alpha 6(RKKG) and alpha 6(RGGR)) were transfected into K562 cel ls. The mutant alpha 6A integrin subunits were expressed in associatio n with endogenous beta 1, at levels comparable to that of the wild-typ e alpha 6A beta 1. A single alpha 6A polypeptide chain (150 kDa) was p recipitated from surface-labeled alpha 6(GKKR), alpha 6(RKKG), and alp ha 6(RGGR) transfectants, while the separate heavy (120 kDa) and light chains (31 or 30 kDa) were precipitated from the wild-type alpha 6(RK KR) transfectant. Thus, a change in the RKKR sequence prevents cleavag e of alpha 6. After activation by the anti-beta 1 stimulatory mAb TS2/ 16 both cleaved and uncleaved alpha 6A beta 1 integrins bound and spre ad on laminin-1. Remarkably, the phorbol ester phorbol 12-myristate 13 -acetate, which activates wild-type alpha 6A beta 1 to bind to laminin -1, did not activate uncleaved alpha 6A beta 1. We conclude that uncle aved alpha 6A beta 1 is capable of ligand binding and transducing outs ide/in signals, like wild type alpha 6A beta 1. However, inside/out si gnaling is affected. It appears that cleavage of alpha 6 is required t o generate the proper conformation in alpha 6 that enables affinity mo dulation of the alpha 6A beta 1 receptor by phorbol 12-myristate 13-ac etate.