title: Major mutations in calf-1 and calf-2 domains of glycoprotein IIb in patients with Glanzmann thrombasthenia enable GPIIb/IIIa complex formation, but impair its transport from the endoplasmic reticulum to the Golgi apparatus.
abstract: The crystal structure of integrin alphavbeta3 comprises 3 regions of contact between alphav and beta3. The main contact on alphav is located in the beta-propeller while calf-1 and calf-2 domains contribute minor interfaces. Whether or not contacts between calf-1 and calf-2 domains of glycoprotein (GP) IIb (alphaIIb) and GPIIIa (beta3) play a role in GPIIb/IIIa complex formation has not been established. In this study we analyzed the effects of 2 naturally occurring mutations in calf-1 and calf-2 domains on GPIIb/IIIa complex formation, its processing, and transport to the cell membrane. The mutations investigated were a deletion-insertion in exon 25 located in calf-2 and an in-frame skipping of exon 20 located in calf-1. Mutated GPIIb cDNAs were cotransfected in baby hamster kidney cells with normal GPIIIa (beta3) cDNA. Analysis by flow cytometry failed to demonstrate detectable amounts of GPIIb or GPIIb/IIIa complex on the surface of cells transfected with each mutation, but immunohistochemical staining revealed their intracellular presence. GPIIb was mainly demonstrable as pro-GPIIb by immunoprecipitation of cell lysates expressing each mutation. Differential immunofluorescence staining of GPIIb and cellular organelles suggested that most altered complexes were located in the endoplasmic reticulum. Homology modeling of normal GPIIb based on the alphavbeta3 crystal structure revealed similar contacts between alphav and beta3 and between alphaIIb and beta3. Introduction of the mutations into the model yielded partial disruption of the normal contacts in the corresponding domains. These data suggest that despite partial disruption of calf-1 or calf-2 domain, GPIIb/IIIa complex is formed but its transport from the endoplasmic reticulum is impaired.
authors.affiliation: Thrombosis and Hemostasis Research Institute, The Chaim Sheba Medical Center, Tel-Hashomer, Israel.
authors.forename: Nurit
authors.initials: N
authors.lastname: Rosenberg
authors.forename: Rivka
authors.initials: R
authors.lastname: Yatuv
authors.forename: Vladimir
authors.initials: V
authors.lastname: Sobolev
authors.forename: Hava
authors.initials: H
authors.lastname: Peretz
authors.forename: Ariella
authors.initials: A
authors.lastname: Zivelin
authors.forename: Uri
authors.initials: U
authors.lastname: Seligsohn
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003460:Crystallization; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D005091:Exons; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D015870:Gene Expression; D006031:Glycosylation; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D039661:Integrin beta3 / Q000378:metabolism*; D007668:Kidney / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D040201:Platelet Membrane Glycoprotein IIb / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D013915:Thrombasthenia / Q000235:genetics*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb