title: Identification of a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets.
abstract: Semaphorin 4D (Sema4D) is a transmembrane protein that supports contact-dependent amplification of platelet activation by collagen before being gradually cleaved by the metalloprotease ADAM17, as we have previously shown. Cleavage releases a soluble 120-kDa exodomain fragment for which receptors exist on platelets and endothelial cells. Here we have examined the mechanism that regulates Sema4D exodomain cleavage. The results show that the membrane-proximal cytoplasmic domain of Sema4D contains a binding site for calmodulin within the polybasic region Arg762-Lys779. Coprecipitation studies show that Sema4D and calmodulin are associated in resting platelets, forming a complex that dissociates upon platelet activation by the agonists that trigger Sema4D cleavage. Inhibiting calmodulin with W7 or introducing a membrane-permeable peptide corresponding to the calmodulin-binding site is sufficient to trigger the dissociation of Sema4D from calmodulin and initiate cleavage. Conversely, deletion of the calmodulin-binding site causes constitutive shedding of Sema4D. These results show that (1) Sema4D is a calmodulin-binding protein with a site of interaction in its membrane-proximal cytoplasmic domain, (2) platelet agonists cause dissociation of the calmodulin-Sema4D complex, and (3) dissociation of the complex is sufficient to trigger ADAM17-dependent cleavage of Sema4D, releasing a bioactive fragment.
authors.affiliation: Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, the First Affiliated Hospital, Soochow University, Suzhou, China.
authors.forename: Peipei
authors.initials: P
authors.lastname: Mou
authors.forename: Zhao
authors.initials: Z
authors.lastname: Zeng
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Liu
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Xin
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Wannemacher
authors.forename: Changgeng
authors.initials: C
authors.lastname: Ruan
authors.forename: Renhao
authors.initials: R
authors.lastname: Li
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Brass
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D016466:CHO Cells; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D015539:Platelet Activation / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D039961:Semaphorins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; C093224:CD100 antigen; D002147:Calmodulin; D004791:Enzyme Inhibitors; D039961:Semaphorins; D013449:Sulfonamides; C017967:W 7
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