The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins.
Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1 class.
Mahimkar
Rajeev M
RM
Department of Medicine, San Francisco Veterans Administration Medical Center/University of California at San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA.
Visaya
Orvin
O
Pollock
Allan S
AS
Lovett
David H
DH
D051722:ADAM Proteins; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D021982:Cell-Matrix Junctions / Q000502:physiology; D019483:Disintegrins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D005982:Glutathione Transferase; D006801:Humans; D039641:Integrin beta Chains / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism*; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D008024:Ligands; D008545:Melanoma / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D010455:Peptides / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins
D019483:Disintegrins; D039641:Integrin beta Chains; D019012:Integrin beta1; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D051722:ADAM Proteins; C098339:ADAM9 protein, human; D008666:Metalloendopeptidases