Clustering and internalization of integrin alphavbeta3 with a tetrameric RGD-synthetic peptide.
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.
Sancey
Lucie
L
INSERM CRI U823, Cibles diagnostiques ou thérapeutiques et vectorisation de drogues dans les cellules tumorales, Institut Albert Bonniot, Grenoble Cedex 9, France.
Lucie
Sancey
S
Garanger
Elisabeth
E
Elisabeth
Garanger
G
Foillard
Stéphanie
S
Stéphanie
Foillard
F
Schoehn
Guy
G
Guy
Schoehn
S
Hurbin
Amandine
A
Amandine
Hurbin
H
Albiges-Rizo
Corinne
C
Corinne
Albiges-Rizo
AR
Boturyn
Didier
D
Didier
Boturyn
B
Souchier
Catherine
C
Catherine
Souchier
S
Grichine
Alexeï
A
Alexeï
Grichine
G
Dumy
Pascal
P
Pascal
Dumy
D
Coll
Jean-Luc
JL
Jean-Luc
Coll
C
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*
D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D010455:Peptides; C047981:arginyl-glycyl-aspartic acid