Cleavage of the alpha6A subunit is essential for activation of the alpha6Abeta1 integrin by phorbol 12-myristate 13-acetate.
The alpha6 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 alpha6 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 alpha6A cDNAs (alpha6GKKR, alpha6RKKG and alpha6RGGR) were transfected into K562 cells. The mutant alpha6A integrin subunits were expressed in association with endogenous beta1, at levels comparable to that of the wild-type alpha6Abeta1. A single alpha6A polypeptide chain (150 kDa) was precipitated from surface-labeled alpha6GKKR, alpha6RKKG, and alpha6-RGGR transfectants, while the separate heavy (120 kDa) and light chains (31 or 30 kDa) were precipitated from the wild-type alpha6RKKR transfectant. Thus, a change in the RKKR sequence prevents cleavage of alpha6. After activation by the anti-beta1 stimulatory mAb TS2/16 both cleaved and uncleaved alpha6Abeta1 integrins bound and spread on laminin-1. Remarkably, the phorbol ester phorbol 12-myristate 13-acetate, which activates wild-type alpha6Abeta1 to bind to laminin-1, did not activate uncleaved alpha6Abeta1. We conclude that uncleaved alpha6Abeta1 is capable of ligand binding and transducing outside/in signals, like wild-type alpha6A-beta1. However, inside/out signaling is affected. It appears that cleavage of alpha6 is required to generate the proper conformation in alpha6 that enables affinity modulation of the alpha6A-beta1 receptor by phorbol 12-myristate 13-acetate.
Delwel
G O
GO
Netherlands Cancer Institute, Division of Cell Biology, Amsterdam, The Netherlands.
Hogervorst
F
F
Sonnenberg
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D006801:Humans; D039121:Integrin alpha6beta1; D016023:Integrins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D007797:Laminin; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D039121:Integrin alpha6beta1; D016023:Integrins; D007797:Laminin; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate