title: Clustering and internalization of integrin alphavbeta3 with a tetrameric RGD-synthetic peptide.
abstract: 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.
authors.affiliation: INSERM CRI U823, Cibles diagnostiques ou thérapeutiques et vectorisation de drogues dans les cellules tumorales, Institut Albert Bonniot, Grenoble Cedex 9, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Sancey
authors.forename: Sancey
authors.initials: S
authors.lastname: Lucie
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Garanger
authors.forename: Garanger
authors.initials: G
authors.lastname: Elisabeth
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Foillard
authors.forename: Foillard
authors.initials: F
authors.lastname: Stéphanie
authors.forename: Guy
authors.initials: G
authors.lastname: Schoehn
authors.forename: Schoehn
authors.initials: S
authors.lastname: Guy
authors.forename: Amandine
authors.initials: A
authors.lastname: Hurbin
authors.forename: Hurbin
authors.initials: H
authors.lastname: Amandine
authors.forename: Corinne
authors.initials: C
authors.lastname: Albiges-Rizo
authors.forename: Albiges-Rizo
authors.initials: AR
authors.lastname: Corinne
authors.forename: Didier
authors.initials: D
authors.lastname: Boturyn
authors.forename: Boturyn
authors.initials: B
authors.lastname: Didier
authors.forename: Catherine
authors.initials: C
authors.lastname: Souchier
authors.forename: Souchier
authors.initials: S
authors.lastname: Catherine
authors.forename: Alexeï
authors.initials: A
authors.lastname: Grichine
authors.forename: Grichine
authors.initials: G
authors.lastname: Alexeï
authors.forename: Pascal
authors.initials: P
authors.lastname: Dumy
authors.forename: Dumy
authors.initials: D
authors.lastname: Pascal
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Coll
authors.forename: Coll
authors.initials: C
authors.lastname: Jean-Luc
mesh_terms: D002460:Cell Line; D036681:Fluorescence Recovery After Photobleaching; D005822:Genetic Vectors / Q000138:chemical synthesis* / Q000737:chemistry*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009842:Oligopeptides / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry*
chemical_list: D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D010455:Peptides; C047981:arginyl-glycyl-aspartic acid