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 biosynthesis in a covalently associated heavy and light chain, To examine the importance of cleavage for the function of the alpha 6 subunit, we introduced mutations in the cDNA encoding the RKKR (876-879) sequence, the presumed cleavage site, in which either one or two basic residues were replaced by glycine. Wild-type and mutant alpha 6A cDNAs (alpha 6(GKKR), alpha 6(RKKG) and alpha 6(RGGR)) 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 the wild-type alpha 6A beta 1. A single alpha 6A polypeptide chain (150 kDa) was precipitated from surface-labeled alpha 6(GKKR), alpha 6(RKKG), and alpha 6(RGGR) transfectants, while the separate heavy (120 kDa) and light chains (31 or 30 kDa) were precipitated from the wild-type alpha 6(RKKR) transfectant. Thus, a change in the RKKR sequence prevents cleavage 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 spread 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 uncleaved alpha 6A beta 1 is capable of ligand binding and transducing outside/in signals, like wild type alpha 6A beta 1. However, inside/out signaling is affected. It appears that cleavage of alpha 6 is required to generate the proper conformation in alpha 6 that enables affinity modulation of the alpha 6A beta 1 receptor by phorbol 12-myristate 13-acetate.