Fibrinogen interaction of CHO cells expressing chimeric alphaIIb/alphavbeta3 integrin.
OBJECTIVE
The molecular mechanisms of the affinity regulation of alphavbeta3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of alphavbeta3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-function within these domains remains unclear.


METHODS
The extracellular and transmembrane domain of alphaIIb was fused to the alphav integrin cytoplasmic domain, and the chimeric alpha subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type beta3 subunit or with 3 mutant beta3 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen.


RESULTS
All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric alpha subunit with the wild-type beta3 subunit, but not those with truncated beta3, could adhere to and spread on immobilized fibrinogen.


CONCLUSIONS
The substitution alphaIIb at the cytoplasmic domain with the alphav cytoplasmic sequence rendered the extracellular alphaIIbbeta3 a constitutively activated conformation for ligands without the need of pinside-outq signals. Our results also indicated that the COOH-terminal sequence of beta3 might play a key role in integrin alphaIIb/alphavbeta3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing alphaIIb/alphavbeta3 have enormous potential for facilitating drug screening for antagonists either to alphavbeta3 intracellular interactions or to alphaIIbbeta3 receptor functions.
Chen
Juan-juan
JJ
Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Su
Xiao-yu
XY
Xi
Xiao-dong
XD
Lin
Li-ping
LP
Ding
Jian
J
Lu
He
H
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics* / Q000502:physiology*; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D039302:Integrin alphaVbeta3; D050506:Mutant Chimeric Proteins; D005340:Fibrinogen