Role of the transmembrane domain of glycoprotein IX in assembly of the glycoprotein Ib–IX complex

Abstract

Summary. Background: The glycoprotein (GP) Ib-IX complex is critically involved in platelet adhesion to von Willebrand factor and in the initial step of platelet activation. How this complex is assembled is not clear. We previously showed that the transmembrane (TM) domains of the GPIba and GPIbb subunits interact and participate in complex assembly. Objectives and methods: Here, we have investigated the role of the TM and cytoplasmic domains of GPIX in assembly of the GPIb-IX complex, by analyzing the mutational effects on complex expression and assembly in transiently transfected Chinese hamster ovary cells. Results: Replacing the cytoplasmic domain of GPIX with a poly-alanine sequence had little effect on surface expression and structural integrity of the GPIb-IX complex. In contrast, replacing the GPIX TM domain (residues 132-153) with a poly-leucine-alanine sequence markedly disrupted complex formation of GPIX with GPIba, interfered with GPIb formation, and decreased surface expression of the host complex. We further analyzed the contributions of a number of GPIX TM residues to complex formation by mutagenesis and found significant roles for Asp135 and several Leu residues. Conclusions: The TM domain, rather than the cytoplasmic domain, of GPIX plays an important role in expression and assembly of the GPIb-IX complex by interacting with its counterparts of GPIb. These TM domains may form a parallel four-helical bundle structure in the complex.

The dimerization interface of the glycoprotein Ibβ transmembrane domain corresponds to polar residues within a leucine zipper motif

“…Specific residues in the TM domain, namely, Gln129 and His139, have been shown to be crucial in the functional assembly of the GP Ib-IX complex, possibly by stabilizing TM-TM associations. 9,13 Our previous studies showed that only Ibb, and not Iba or IX, was able to self-associate in the SDS detergent micelle and E. coli cell membrane. 13 However, the specific residues or motifs that determine the association of the Ibb TM domain have not been thoroughly examined.…”

“…8 Further studies showed that TM domain associations of Iba, Ibb, and IX domain favorably position the membrane-proximal Cys residues for disulfide bond formation, and this supports the ab 2 configuration. 5,9 All these observations suggest that the TM domains of the GP Ib-IX complex are responsible for functionally important associations of the GP Ib-IX complex in membranes.…”

Single novel mutation in transmembrane region of glycoprotein (GP) IX affects GP Ib‐ IX complex expression and causes Bernard–Soulier syndrome

“…The 1388G>A; Trp134 fi stop mutation deleted nearly all the TM region and all of the intracellular residues of the GP IX protein. Given that deletion of the TM region is known to reduce the GP Ib-IX complex's surface expression on platelets (Luo et al, 2007), the 1388G>A; Trp134 fi stop muation caused the BSS in our patient.…”

“…Recently, studies in transfected cells have disclosed the important role and molecular basis of GP IX's TM domain for the assembly and expression of GP Ib-IX-V complex on platelets. Notably, Asp153 (interacting with Gln129 of GPIbb) and several leucine residues (Leu136, Leu138, Leu148, Leu149, Leu153), which form leucine-zipper-like sequences and thus interact with GP Ibb, are important for normal assembly and surface expression of the GP Ib-IX complex (Luo et al, 2007). Based on this knowledge, it is easy to understand why the mutation Trp134 fi stop is responsible for BSS.…”

“…GP IX is composed of a signal peptide of 16 amino acids and the mature proteins of 161 amino acids, the latter contains an N-terminal extracellular domain (1-131aa), TM domain (132-153aa), and intracellular residues (154-161aa) (Luo et al, 2007). Recently, studies in transfected cells have disclosed the important role and molecular basis of GP IX's TM domain for the assembly and expression of GP Ib-IX-V complex on platelets.…”