Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex

Abstract

Engagement of platelet membrane glycoprotein (GP) Ib-IX-V by von Willebrand factor triggers Ca ؉؉ -dependent activation of ␣IIb␤3, resulting in (patho)physiological thrombus formation. It is demonstrated here that the cytoplasmic domain of GPIb-IX-V associates with cytosolic calmodulin. First, an anti-GPIb␣ antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following platelet stimulation, calmodulin dissociated from GPIb-IX and, like the GPIb-IXassociated proteins 14-3-3 and p85, redistributed to the activated cytoskeleton. Second, a synthetic peptide based on the cytoplasmic sequence of GPIb␤, R149-L167 (single-letter amino acid codes), affinity-isolated calmodulin from platelet cytosol in the presence of Ca ؉؉ as confirmed by comigration with bovine calmodulin on sodium dodecyl sulfate-polyacrylamide gels, by sequence analysis, and by immunoreactivity with the use of an anticalmodulin antibody. The membraneproximal GPIb␤ sequence was analogous to a previously reported calmodulinbinding sequence in the leukocyte adhesion receptor, L-selectin. In addition, the cytoplasmic sequence of GPV, K529-G544, was analogous to a calmodulinbinding IQ motif within the ␣1c subunit of L-type Ca ؉؉ channels. Calmodulin coimmunoprecipitated with GPV from resting platelet lysates, but was dissociated in stimulated platelets. A GPV-related synthetic peptide also bound calmodulin and induced a Ca ؉؉ -dependent shift on nondenaturing gels. Together, these results suggest separate regions of GPIb-IX-V can directly bind calmodulin, and this novel interaction potentially regulates aspects of GPIb-IX-V-dependent platelet activation. IntroductionThe platelet membrane glycoprotein (GP) Ib-IX-V complex binds to the adhesive ligand von Willebrand factor (vWF), an interaction that initiates platelet adhesion and aggregation at high-shear stress. [1][2][3][4] Adhered platelets become activated, with elevation of cytosolic Ca ϩϩ and triggering of signaling pathways, leading to cytoskeletal rearrangements and activation of ␣IIb␤3 integrin (GPIIb-IIIa), which binds to fibrinogen or vWF, resulting in platelet aggregation. [5][6][7] The pathway by which ligand binding to GPIb-IX-V leads to ␣IIb␤3 activation and thrombus formation is poorly understood, although delineation of early signaling events mediated by GPIb-IX-V offers potential targets for therapeutic intervention.The GPIb-IX-V complex consists of 4 membrane-spanning glycoproteins. GPIb␣ is disulfide-linked to GPIb␤, forming GPIb, which is noncovalently associated with GPIX in a 1:1 complex. GPIb-IX-V is composed of GPIb-IX and GPV in a 2:1 ratio. 3 In extracts of platelets in the presence of Triton X-100 detergent, GPIb-IX remains a complex, while GPV is dissociated. 8,9 The extracellular, N-terminal 282 residues of GPIb␣ contain the vWF binding site. 3,10 Recent studies have identified signaling molecules associated with the cytoplasmic domain of GPIb-IX-V that appear to be important for the function of the receptor. One of these molecules is 14-3-3, which direc...

A Mitogen-activated Protein Kinase-dependent Signaling Pathway in the Activation of Platelet Integrin αIIbβ3

“…7) (43)(44)(45)(46). In addition, GPIb-IX is associated with several intracellular signaling proteins including 14-3-3 (47,48), phosphatidylinositol 3-kinase (via 14-3-3) (49), and calmodulin (50). Although the roles of these proteins in GPIb-IX signaling are still unclear, it is possible that these proteins are also involved in GPIb-IX-mediated signaling.…”

Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

“…14-3-3 has been shown to be involved in GPIb-IX-mediated activation of the integrin ␣ IIb ␤ 3 (6). The cytoplasmic domain of GPIb␤ has been found to interact with calmodulin between residues 149 and 167 (16). GPIb␤ has also been found to interact with 14-3-3 (17,18) and is phosphorylated at Ser 166 by PKA (19 -21).…”

Platelet Interactions in Thrombosis

“…Fourth, an antiGPIba monoclonal antibody, SZ2, which maps to the anionic/ sulphated sequence of GPIba inhibits collagen-induced platelet aggregation mediated by GPVI (1). Fifth, both GPIb-IX-V (GPIbb and GPV) and GPVI directly associate via their cytoplasmic domains with calmodulin, interactions that dissociate following platelet activation (24,35,36). It is conceivable calmodulin association maintains an adhesive complex on resting platelets.…”

“…(12,15). Immediately N-terminal to the 14-3-3x-binding site of GPIbb is a positively-charged, membrane-proximal sequence that binds calmodulin (24). A different calmodulin-binding sequence is within the short, *16-residue cytoplasmic tail of GPV.…”

“…A different calmodulin-binding sequence is within the short, *16-residue cytoplasmic tail of GPV. Both 14-3-3x and calmodulin dissociate from GPIb-IX-V following platelet activation (15,24). Reconstitution of GPIb-dependent activation of aIIbb3 in mammalian cell lines has revealed that an intact 14-3-3x-binding site on GPIba is essential for this process, and that activation of ERK-1/2 kinase is a likely downstream mediator (25).…”