Figure 5.
Figure 5. Normal and mutant αIIb subunits associate with calnexin. (A) Whole-cell lysates of cells either transiently or stably expressing normal αIIbβ3, VFαIIb plus β3, or αIIb alone were immunoprecipitated with antibodies against αIIb, subjected to SDS-PAGE, and then immunoblotted with mAbs against αIIb (PMI-1, top left panel, or H-160, top right panel) or calnexin (AF8, bottom left panel, or SPA-865, bottom right panel). Calnexin was coprecipitated from cells expressing normal αIIbβ3, either transiently or stably, VFαIIb plus β3, and cells expressing αIIb alone, but not from mock (vector-only)–transfected cells. As a loading control, equivalent amounts of whole-cell lysate from each cell type were subjected to SDS-PAGE and immunoblotted with anticalnexin, revealing that equal amounts of total calnexin had been loaded (data not shown). (B,C) Pulse-chase analysis demonstrated a markedly prolonged t1/2 for pro-N15QαIIb (17 ± 2 hours) as compared with normal pro-αIIb (5 ± 2 hours, n = 3, P < .001), and failure of pro-N15QαIIb to undergo normal processing to mature N15QαIIb or to form the N15QαIIb-β3 complex normally. (D) The top panel shows the sequence alignment of the first 60 amino acids of human αIIb with αIIb from mouse, rat, pig, horse, rabbit, and dog. The bottom panel shows the sequence alignment of the corresponding residues of human αV, α8, α5, α3, α7, and α4. N-linked glycosylation consensus sequences at position 15 are highlighted in yellow, and those at other positions are in cyan. Residues comprising β strands are indicated in magenta. (E) Location of N15 in the αIIbβ propeller, and the relative location of N45 in αV. Propeller blades are numbered 1 through 7 and the Cap domain is indicated. In the crystal structures of both subunits, residues at position 15 are located at the top outer corner of the first propeller blade, whereas residues at approximately position 45 lie at the apex of the next upward-facing loop of the same blade, thus occupying a position adjacent to residue 15 on the propeller's surface.23,24

Normal and mutant αIIb subunits associate with calnexin. (A) Whole-cell lysates of cells either transiently or stably expressing normal αIIbβ3, VFαIIb plus β3, or αIIb alone were immunoprecipitated with antibodies against αIIb, subjected to SDS-PAGE, and then immunoblotted with mAbs against αIIb (PMI-1, top left panel, or H-160, top right panel) or calnexin (AF8, bottom left panel, or SPA-865, bottom right panel). Calnexin was coprecipitated from cells expressing normal αIIbβ3, either transiently or stably, VFαIIb plus β3, and cells expressing αIIb alone, but not from mock (vector-only)–transfected cells. As a loading control, equivalent amounts of whole-cell lysate from each cell type were subjected to SDS-PAGE and immunoblotted with anticalnexin, revealing that equal amounts of total calnexin had been loaded (data not shown). (B,C) Pulse-chase analysis demonstrated a markedly prolonged t1/2 for pro-N15QαIIb (17 ± 2 hours) as compared with normal pro-αIIb (5 ± 2 hours, n = 3, P < .001), and failure of pro-N15QαIIb to undergo normal processing to mature N15QαIIb or to form the N15QαIIb-β3 complex normally. (D) The top panel shows the sequence alignment of the first 60 amino acids of human αIIb with αIIb from mouse, rat, pig, horse, rabbit, and dog. The bottom panel shows the sequence alignment of the corresponding residues of human αV, α8, α5, α3, α7, and α4. N-linked glycosylation consensus sequences at position 15 are highlighted in yellow, and those at other positions are in cyan. Residues comprising β strands are indicated in magenta. (E) Location of N15 in the αIIbβ propeller, and the relative location of N45 in αV. Propeller blades are numbered 1 through 7 and the Cap domain is indicated. In the crystal structures of both subunits, residues at position 15 are located at the top outer corner of the first propeller blade, whereas residues at approximately position 45 lie at the apex of the next upward-facing loop of the same blade, thus occupying a position adjacent to residue 15 on the propeller's surface.23,24 

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