Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex.
Engagement of platelet membrane glycoprotein (GP) Ib-IX-V by von Willebrand factor triggers Ca(++)-dependent activation of alphaIIbbeta3, 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-GPIbalpha antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following platelet stimulation, calmodulin dissociated from GPIb-IX and, like the GPIb-IX-associated proteins 14-3-3zeta and p85, redistributed to the activated cytoskeleton. Second, a synthetic peptide based on the cytoplasmic sequence of GPIbbeta, 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 membrane-proximal GPIbbeta sequence was analogous to a previously reported calmodulin-binding sequence in the leukocyte adhesion receptor, L-selectin. In addition, the cytoplasmic sequence of GPV, K529-G544, was analogous to a calmodulin-binding IQ motif within the alpha1c 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. (Blood. 2001;98:681-687)
Andrews
R K
RK
Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Research Institute, St. Kilda Road Central, Melbourne, Victoria, Australia. rkandrews@hotmail.com
Munday
A D
AD
Mitchell
C A
CA
Berndt
M C
MC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism / Q000648:ultrastructure; D002147:Calmodulin / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex