Glycoprotein Ibβ is the only phosphorylated major membrane glycoprotein in human platelets

Abstract

Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.

The glycoprotein Ib complex of human blood platelets

“…Kalomiris et al [23] reported that GPIbfl is a transmembrane protein with a reactive endofacial sulfhydryl group. Wyler et al [22] showed that the / 3 subunit of GPIb contains a phosphorylation site. While GPIbp and GP17 are similar in their molecular mass and are labeled similarly with carbohydrate-specific labeling methods, we were able to demonstrate that polyclonal rabbit antibodies against these two glycoproteins do not cross-react.…”

“…Fox et al [20] and Okita et al [21] reported that GPIb is linked via actin-binding protein to the cytoskeleton in resting platelets. Wyler et al [22] have shown that the GPIbg subunit is phosphorylated; Kalomiris et al [23] reported labeling of the p subunit by membrane-permeable fluorescent reagents which bind to reactive thiol groups. Methods for the purification of intact GPIb have only recently been established and much remains to be discovered about its structure.…”

“…Gels were stained with silver. Numbers on the right indicate apparent molecular mass in kDa intact GPIb prior to the analysis [22]. However, the remnant of the a chain could not be detected and its molecular mass was estimated from the difference between that of the nonreduced form of truncated GPIb and the P subunit after reduction.…”

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

“…In nonactivated platelets, GPIb is complexed to actin-binding protein and thereby to the cytoskeleton, via the intracytoplasmic segment of the alpha chain (3). The role of GPIb3 is unknown, although it is the major platelet membrane protein to be phosphorylated when resting platelets are incubated with [32P]phosphate (4). This phosphorylation, thought to occur at Serl66 in the mature platelet protein, is present under conditions that increase intracellular cAMP and may contribute to the inhibitory actions of cAMP by inhibiting collagen-induced polymerization of actin (5).…”

The Cytoplasmic Domain of the Platelet Glycoprotein Ibα Is Phosphorylated at Serine 609

“…Previously, phosphorylation of platelet membrane proteins has been studied (30,36). The only serine/threonine-phosphorylated major membrane glycoprotein identified was GPIb␤ (30). GPIb␤ is phosphorylated at Thr 166 when platelets are stimulated with agents that enhance intracellular cAMP level (36,37).…”