SERINE-752 IN THE CYTOPLASMIC DOMAIN OF THE BETA-3 INTEGRIN SUBUNIT IS NOT REQUIRED FOR ALPHA-V-BETA-3 POSTRECEPTOR SIGNALING EVENTS

A naturally occurring point mutation (Ser(752)pro substitution) in the beta subunit cytoplasmic domain of the platelet fibrinogen receptor G PIIb-IIIa (integrin alpha IIb beta 3), causing Glanzmann's thrombasthe nia, has been shown to abrogate bidirectional transmembrane signaling of GPIIb-IIIa when expressed in heterologous cells (Chen YP, 1994, Blo od 84, 1857-1865). As the vitronectin receptor alpha v beta 3 constitu tively mediates cell attachment to RGD containing extracellular matrix proteins, the purpose of this study was to explore the regulatory rol e of Ser(752) in alpha v beta 3 vitronectin receptor function, by cotr ansfecting recombinant human cry cDNA together with human beta 3 mutan t cDNA into Chinese hamster ovary (CHO) cells. CHO cells expressing wi ld type human alpha v beta 3 acquired the ability to attach and spread on fibrinogen and von Willebrand factor, in contrast to non transfect ed CHO cells that only bound to vitronectin and fibronectin. Overexpre ssion of a truncated recombinant beta 3 subunit (beta 3 Delta 744) gen erated alpha v(hamster)beta 3(human) chimers that mediated attachment but lost the ability to promote cell spreading on vitronectin, von Wil lebrand factor and fibrinogen, and to concentrate in focal contact sit es, demonstrating a negative effect of beta 3 Delta 744 on alpha v bet a 3 dependent postreceptor occupancy events. Transfection of beta 3Ser (752)pro reproduced the same negative effect as beta 3 Delta 744, wher eas beta 3Ser(752)Ala restored normal receptor function by allowing pr onounced attachment and spreading on fibrinogen and von Willebrand fac tor. Our results provide evidence that (1) the C-terminal cytoplasmic domain of beta 3 (amino acids 744-762) is essential for alpha v beta 3 integrin postreceptor occupancy events; (2) within this domain, the S er(752)pro mutation affects alpha v beta 3 postreceptor occupancy even ts by preventing cell spreading and focal contact localization; (3) th e defective receptor function of the vitronectin receptor alpha v beta 3 is due to the presence of pro(752), rather than the absence of Ser( 752), as a Ser to Ala substitution at position 752 restores normal bet a 3 integrin cell spreading and adhesive plaque formation.