title: Competition between normal [674C] and mutant [674R] subunits: role of the molecular chaperone BiP in the processing of GPIIb-IIIa complexes.
abstract: This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surface.
authors: Department of Pathophysiology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
authors: E G
authors: EG
authors: Arias-Salgado
authors: N
authors: N
authors: Butta
authors: C
authors: C
authors: González-Manchón
authors: S
authors: S
authors: Larrucea
authors: M S
authors: MS
authors: Ayuso
authors: R
authors: R
authors: Parrilla
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D006224:Cricetinae; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013915:Thrombasthenia / Q000097:blood / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D019039:Platelet Glycoprotein GPIIb-IIIa Complex