Although the urokinase receptor (uPAR) binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen bound to VN fragments containing the heparin-binding domain, indicating that this domain was functionally active in the recombinant peptides. However, no significant binding was detected when uPAR was incubated with this domain, and neither heparin nor plasminogen competed with it for binding to VN. In fact, uPAR only bound to fragments containing the somatomedin B (SMB) domain, and monoclonal antibodies (mAbs) that bind to this domain competed with uPAR for binding to VN. Monoclonal antibody 8E6 also inhibited uPAR binding to VN, and this mAb was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site by acid treatment eliminated mAb 8E6 binding but had no effect on uPAR binding. Thus, there appears to be a single binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized by mAb 8E6. Inhibition of uPAR binding to VN by mAb 8E6 probably results from steric hindrance. Vitronectin (VN)1 is a 75-kDa adhesive glycoprotein. It circulates in blood in a monomeric ("closed," "native") form, but is converted into a multimeric ("extended," "opened," "denatured") form when incorporated into the extracellular matrix or treated with urea (1, 2). The extended form of VN binds to specific receptors on cells (3, 4) and to various other molecules such as the C5b-9 complement complex (5), the thrombin-antithrombin III complex (6, 7), plasminogen activator inhibitor 1 (PAI-1) (8 -10), uPAR (11), heparin (1, 2, 12, 13), collagen (14 -16), plasminogen (13, 17), and ␤-endorphin (18). These interactions not only promote the attachment, spreading, and growth of cells (19 -21) but also influence the coagulation, fibrinolytic, and complement systems (22,23).Although a number of investigators have attempted to identify the binding site(s) in VN for these molecules, the literature remains somewhat controversial. For example, three different regions of the VN molecule have been proposed to contain the binding sites for uPAR and PAI-1. The first region, the somatomedin B (SMB) domain (residues 1-44) was identified from direct binding studies (9, 24 -27) and from studies showing that soluble SMB competes with uPAR and PAI-1 for binding to denatured VN (9). The second region in VN that has been implicated in uPAR and PAI-1 binding is the heparin binding (HB) domain (residues 348 -370). Thus, synthetic peptides from this domain interfere with both uPAR (21) and PAI-1 (28) binding to VN. Moreover, mAb 8E6 (which has been mapped to the HB domain (Ref. 13)) also inhibits the binding of PAI-1 to VN. Although a third region in VN (residues 115-121) was shown to have weak PAI-...

Interaction with Fe(III) induces the reversible conformational switch of the extramembrane segment in the artificial receptor channel, which is transmitted into membranes as an increase in channel current (ion flux).

No abstract

68Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting 68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle 67Ga, with the previously described 67Ga-DOTA complex conjugated bisphosphonate, 67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5±1.5, 15.1±2.6, and 12.8±1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp)11 and 67Ga-DOTA-(Asp)14 were comparable with those of 67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of 68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.