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 (Ser752Pro substitution) in the beta subunit cytoplasmic domain of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3), causing Glanzmann's thrombasthenia, has been shown to abrogate bidirectional transmembrane signaling of GPIIb-IIIa when expressed in heterologous cells (Chen YP, 1994, Blood 84, 1857-1865). As the vitronectin receptor alpha v beta 3 constitutively mediates cell attachment to RGD containing extracellular matrix proteins, the purpose of this study was to explore the regulatory role of Ser752 in alpha v beta 3 vitronectin receptor function, by cotransfecting recombinant human alpha v cDNA together with human beta 3 mutant cDNA into Chinese hamster ovary (CHO) cells. CHO cells expressing wild type human alpha v beta 3 acquired the ability to attach and spread on fibrinogen and von Willebrand factor, in contrast to non transfected CHO cells that only bound to vitronectin and fibronectin. Overexpression of a truncated recombinant beta 3 subunit (beta 3 delta 744) generated alpha v (hamster) beta 3 (human) chimers that mediated attachment but lost the ability to promote cell spreading on vitronectin, von Willebrand factor and fibrinogen, and to concentrate in focal contact sites, demonstrating a negative effect of beta 3 delta 744 on alpha v beta 3 dependent postreceptor occupancy events. Transfection of beta 3Ser752Pro reproduced the same negative effect as beta 3 delta 744, whereas beta 3Ser752Ala restored normal receptor function by allowing pronounced attachment and spreading on fibrinogen and von Willebrand factor. 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 Ser752Pro mutation affects alpha v beta 3 postreceptor occupancy events by preventing cell spreading and focal contact localization; (3) the defective receptor function of the vitronectin receptor alpha v beta 3 is due to the presence of Pro752, rather than the absence of Ser752, as a Ser to Ala substitution at position 752 restores normal beta 3 integrin cell spreading and adhesive plaque formation.
Kieffer
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Laboratoire Franco-Luxembourgeois de Recherche Biomédicale, Center Universitaire Luxembourg, Grand-Duchy. Kieffer@cu.lu
Melchior
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Guinet
J M
JM
Michels
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Gouon
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Bron
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D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology; D006224:Cricetinae; D003599:Cytoskeleton / Q000502:physiology / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D039661:Integrin beta3; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D019030:Receptors, Vitronectin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013915:Thrombasthenia / Q000235:genetics; D014162:Transfection; D019096:Vitronectin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D016326:Extracellular Matrix Proteins; D039661:Integrin beta3; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; D019096:Vitronectin; D014841:von Willebrand Factor; D012694:Serine; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D011392:Proline; D000409:Alanine