Integrin clustering mechanisms explored with a soluble alphaIIbbeta3 ectodomain construct.
The purpose of this study was to test the hypothesis that residues critical for ligand- and temperature-induced clustering of integrin alphaIIbbeta3 are present on its extracellular domain. Sucrose density gradient sedimentation was used to examine the effects of ligand-mimetic peptides and physiological temperature on the oligomeric state of a soluble recombinant ectodomain variant of the alphaIIbbeta3 integrin, alphaIIbDelta962beta3Delta692, and its full-length counterpart. Both the ectodomain construct, isolated from High Five insect cell culture supernatants, and alphaIIbbeta3, isolated from human blood platelets, exhibited similar weight-average sedimentation coefficients at 23 degrees C, in the absence and presence of the ligand-mimetic peptide eptifibatide. These observations indicate that alphaIIbbeta3's ectodomain exhibits a similar extended conformation in both its free and ligand-bound states. Oligomerization was examined by incubation of both alphaIIbDelta962beta3Delta692 and full-length receptors at 37 degrees C, in the presence or absence of ligand-mimetic. Minimal oligomerization was observed with alphaIIbDelta962beta3Delta692. In contrast, full-length alphaIIbbeta3 exhibited substantial temperature-induced increases in its distribution of sedimenting species, indicative of thermal aggregation. These observations suggest that optimum oligomerization requires the participation of the integrin's transmembrane and cytoplasmic regions. In vivo, clustering of ligand-bound integrins may enhance signaling by increasing the local concentration of intracellular integrin-associated proteins.
Hantgan
R R
RR
Department Biochemistry, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1019, USA. rhantgan@wfubmc.edu
Gibbs
W
W
Stahle
M C
MC
Aster
R H
RH
Peterson
J A
JA
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D007313:Insecta; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012995:Solubility; D012996:Solutions; D013696:Temperature
D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D012996:Solutions