Transmembrane and Trans-subunit Regulation of Ectodomain Shedding of Platelet Glycoprotein Ibα

Abstract

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 GPIb␣, 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 GPIb␣ sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIb␣, GPIb␤, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIb␤ is critical for ADAM17-dependent shedding of GPIb␣ induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIb␣ and GPIb␤ subunits further lessened the restraint of GPIb␤ on GPIb␣ shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIb␤ cytoplasmic domain disrupted calmodulin binding to GPIb␤ but had little effect on GPIb␣ shedding. Analysis of induction of GPIb␣ shedding by membrane-permeable GPIb␤-derived peptides implicated the association of GPIb␤ with an unidentified intracellular protein in mediating regulation of GPIb␣ shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.A large number of transmembrane proteins expressed on the cell surface undergo ectodomain shedding, a process in which the protein is cleaved at a site close to the outer surface of the cell membrane and subsequently its ectodomain released from the cell (1). Because ectodomain shedding plays a vital role in various cellular processes, including cell growth and proliferation, mammalian development, cell migration, and inflammation, its aberration often leads to disease states including cancer (2-5). Ectodomain shedding is in essence an enzyme-driven proteolytic reaction; therefore, in general there are two ways to regulate it. One way is to modulate the activity of the enzyme that is often referred to as sheddase; the other is to modulate the accessibility of the shedding substrate to the sheddase. Most sheddases belong to the ADAM (a disintegrin and metalloprotease) 2 protease family within the metzincin superfamily (1). Although the structure, function, and regulation of ADAMs have been extensively studied (6 -8), mechanisms modulating the accessibility of shedding substrates have not been well explored.In addition to extracellular signals such as ligand binding to the shedding substrate (9, 10), intracellular signals can also induce shedding of a protein without affecting the catalytic activity of the sheddase. The first reported example of this mechanism is the induction of shedding of L-selectin by calmodulin (CaM) inhibitors (11). CaM co-immunoprecipitates with L-selectin from cell lysates and binds directly to the isolated cytoplasmic domain of L-selectin. Treating L-selectin-expressing cells with trifluoperazine, a CaM inhibitor, causes dissociation of CaM...

Interaction of Calmodulin with l-Selectin at the Membrane Interface: Implication on the Regulation of l-Selectin Shedding

“…18–23 However, there have been observations that do not seem consistent with the CaM hypothesis. 18; 25 It also remains unclear how CaM binding to the cytoplasmic domain of a shedding substrate can influence its proteolytic cleavage on the other side of the membrane. In this study we have characterized the association of CaM with CLS, a peptide containing both transmembrane and cytoplasmic domains of L-selectin, at the surface of a membrane bilayer.…”

“…18 Similarly, while shedding of the GPIbα subunit in the GPIb-IX receptor complex can be stimulated by CaM inhibitors, a mutation in the cytoplasmic domain of the complex that abolishes CaM association does not lead to elevated shedding of GPIbα. 25 …”

Identification of a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets

“…W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) is a membrane-permeable compound that has been shown to induce shedding of other platelet membrane proteins. 33,36,43,44 It is thought to do this either by causing a change in the structure of calmodulin 45 or by occupying the hydrophobic pocket found in calmodulin. 46 In the experiments summarized in Figure 2A, platelets were incubated with 150 mM W7 for up to 60 minutes.…”

“…Shedding of both proteins involves the association of their intracellular domains with calmodulin, [32][33][34][35] as does the shedding of PECAM-1 (CD31). 36 It is not clear, however, that this is a general mechanism for regulating shedding in platelets.…”

Proteolytic processing of platelet receptors

“…As the process is largely driven by metalloproteinases, control of receptor cleavage events is likely to be provided either by direct inhibition of the catalytic process or by controlling access of the enzyme to the substrate. In the case of GPIbα, roles for a membrane‐proximal region of the GPIbβ cytoplasmic domain and a 28‐amino acid mechanosensory domain within the extracellular juxtamembrane region of GPIbα in maintenance of stable surface levels of the GPIbα subunit have been identified. Both of these regions regulate the availability of the ADAM17 cleavage site within GPIbα to metalloproteases such as ADAM17 and so aid in control of GPIbα levels.…”