Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion.
Vitronectin (VN), a major cell adhesion protein, is found in plasma and in the extracellular matrix. At least three distinct cell surface receptors for vitronectin belonging to the integrin superfamily have been identified in normal and neoplastic cells. Many cell adhesion ligands, including vitronectin, contain an Arg-Gly-Asp (RGD) sequence mediating, in part, the ligand-receptor interaction. These ligands bind different integrins with varying specificity and affinity. The mechanism of receptor specificity remains controversial. To determine the role of the RGD sequence in receptor specificity, we amplified the cDNA for human vitronectin from a liver cDNA library and generated two separate mutants by utilizing site-directed mutagenesis resulting in aspartic acid (Asp47) to glutamic acid (Glu47) substitution and glycine (Gly46) to alanine (Ala46) substitution. The mammalian expression vector, pZEM229R, was used to transfect baby hamster kidney cells which secreted recombinant proteins into the supernatant. All recombinant proteins were isolated by heparin-agarose chromatography and tested for interaction with three known vitronectin receptors, namely, alpha IIIb beta 3 on thrombin-activated platelets, alpha v beta 3 on human umbilical vein endothelial cells and alpha v beta 5 on Panc-1 cells. Recombinant wild-type vitronectin behaved in a fashion similar to plasma-derived vitronectin. Both the RGE-VN and RAD-VN recombinant mutant proteins showed complete loss of cell adhesion activity, regardless of the receptor. These results confirm the essential and central role of the RGD sequence in vitronectin for cell adhesion. This expression system allows further structure/function analysis of vitronectin.
Cherny
R C
RC
Department of Medicine, University of Washington, Seattle 98195.
Honan
M A
MA
Thiagarajan
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004730:Endothelium, Vascular / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D012516:Osteosarcoma; D010190:Pancreatic Neoplasms; D016133:Polymerase Chain Reaction / Q000379:methods; D016024:Receptors, Cytoadhesin / Q000378:metabolism; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D019096:Vitronectin
D006023:Glycoproteins; D009838:Oligodeoxyribonucleotides; D016024:Receptors, Cytoadhesin; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; D019096:Vitronectin; D004247:DNA; D013917:Thrombin