Analysis of the alpha4beta1 integrin-osteopontin interaction.
The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.
Barry
S T
ST
Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom. stb38557@glaxowellcome.co.uk
Ludbrook
S B
SB
Murrison
E
E
Horgan
C M
CM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002874:Chromosome Mapping; D015870:Gene Expression; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D053495:Osteopontin; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*
D039041:Integrin alpha4beta1; D016023:Integrins; D016201:Receptors, Lymphocyte Homing; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin