title: Ca2+ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin alpha v beta 3.
abstract: Osteopontin (OPN) is an extracellular matrix protein that supports osteoclast adhesion to the bone by binding to integrin alpha v beta 3. We measured the binding between OPN and integrin alpha v beta 3 with recombinant human OPN and the urinary form of human OPN, uropontin. Recombinant OPN was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and cleaved from glutathione S-transferase with Factor Xa. The mass of this form of OPN (rOP27) is 27,046 Da. rOP27 is truncated at arginine residue 228, 69 amino acids short of the native carboxyl terminus. Uropontin and rOP27 support RGD-dependent cell adhesion and to bind purified integrin alpha v beta 3 with similar affinities. Further study showed that OPN is the only known naturally occurring RGD-containing protein with a much greater affinity for alpha v beta 3 than for the platelet integrin alpha IIb beta 3. Most importantly, we find that physiologic levels of Ca2+ block cell adhesion to OPN. Measurement of binding constants between rOPN and purified integrin alpha v beta 3 with surface plasmon resonance showed that the affinity between rOPN and alpha v beta 3 is 26-fold lower in Ca2+ (Kd = 1.1 x 10(-8) M) than in Mn2+ (Kd = 4.3 x 10(-10) M) and 9-fold lower than in Mg2+ (Kd = 1.3 x 10(-9) M). In bone, the resorbing osteoclast generates elevated levels of extracellular Ca2+, therefore the findings presented here suggest a previously unappreciated mechanism for the modulation of bone resorption by extracellular Ca2+.
authors.affiliation: Department of Vascular Biology (VB-1), Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: D D
authors.initials: DD
authors.lastname: Hu
authors.forename: J R
authors.initials: JR
authors.lastname: Hoyer
authors.forename: J W
authors.initials: JW
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001862:Bone Resorption / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D053495:Osteopontin; D011485:Protein Binding; D016024:Receptors, Cytoadhesin / Q000378:metabolism*; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins / Q000235:genetics; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D016023:Integrins; D016024:Receptors, Cytoadhesin; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D002118:Calcium