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Integrin alpha(v)beta(3) is overexpressed on neoendothelial cells and frequently on tumor cells. We have developed a peptide-like scaffold (regioselectively addressable functionalized template, RAFT), which holds four cyclo(-RGDfK-) (cRGD) motifs and proved that this molecule (called regioselectively addressable functionalized template-arginine-glycine-aspartic acid, RAFT-RGD) targets integrin alpha(v)beta(3) in vitro and in vivo. Using fluorescence correlation spectroscopy (FCS), we measured the constant of affinity (K(D)) of the RAFT-RGD for purified integrins. K(D) values rose from 3.87 nmol/l for RAFT-RGD to 41.70 nmol/l for cyclo(-RGDfK-). In addition, RAFT-RGD inhibited alpha(v)beta(3) lateral mobility in the cell membrane, probably due to the formation of integrin clusters as demonstrated by fluorescence recovery after photobleaching (FRAP). This was confirmed by electronic microscopy data, which established the formation of molecular complexes containing two integrins in the presence of RAFT-RGD but not cRGD or regioselectively addressable functionalized template-arginine-alanine- aspartic acid (RAFT-RAD). Using an enzyme-linked immunosorbent assay (ELISA), we proved that 1 micromol/l RAFT-RGD increased by 79% alpha(v)beta(3) internalization via clathrin-coated vesicles. Conversely, cRGD was internalized without modifying alpha(v)beta(3) internalization. Although RGD has been known for >20 years, this is the first study to formerly establish the relationships among multimeric presentation, increased affinity, and subsequent integrin-mediated cointernalization. These results strongly support the rationale for using multimeric RGD-peptides as targeting vectors for imaging, diagnosis, or therapy of cancers.