Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.
Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbα, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbα sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIbα, GPIbβ, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIbβ is critical for ADAM17-dependent shedding of GPIbα induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIbα and GPIbβ subunits further lessened the restraint of GPIbβ on GPIbα shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIbβ cytoplasmic domain disrupted calmodulin binding to GPIbβ but had little effect on GPIbα shedding. Analysis of induction of GPIbα shedding by membrane-permeable GPIbβ-derived peptides implicated the association of GPIbβ with an unidentified intracellular protein in mediating regulation of GPIbα shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.
Mo
Xi
X
Department of Biochemistry and Molecular Biology, Center for Membrane Biology, University of Texas Health Science Center, Houston, Texas 77030, USA.
Nguyen
Nam X
NX
Mu
Fi-Tjen
FT
Yang
Wenjun
W
Luo
Shi-Zhong
SZ
Fan
Huizhou
H
Andrews
Robert K
RK
Berndt
Michael C
MC
Li
Renhao
R
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D016296:Mutagenesis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D019038:Platelet Glycoprotein GPIb-IX Complex; D021122:Protein Subunits; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human