Competition between normal [674C] and mutant [674R]GPIIb subunits: role ofthe molecular chaperone BiP in the processing of GPIIb-IIIa complexes

This work aimed at investigating the function of the [C674R] mutation in GP IIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individua ls heterozygous for this mutation show a platelet GPIIb-IIIa content approx imately 30% of normal controls, which is less than expected from one normal functioning allele, Coexpression of normal [674C]GPIIb and mutant [674R]GP IIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhib ition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association an d/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 l ess efficient subunit conformation. The mutant [674R]GPIIb but not normal G PIIb was found associated with the endoplasmic reticulum chaperone BiP in t ransiently 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 dit hiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardatio n of the secretory pathway, most likely related to its binding to the molec ular chaperone BiP, with the result of a defective number of functional GPI Ib-IIIa receptors in the cell surface. (Blood, 2001;97: 2640-2647) (C) 2001 by The American Society of Hematology.