Effect of mutagenesis of GPIIb amino acid 273 on the expression and conformation of the platelet integrin GPIIb-IIIa.
A G273D mutation immediately proximal to the first calcium binding domain of platelet GPIIb impairs the export of GPIIb-IIIa heterodimers to the platelet surface. To examine how this mutation might alter the structure of GPIIb, G273 was replaced by other amino acids and the resulting mutants were coexpressed with GPIIIa in COS-1 cells. Although replacement with Ala or Val had no effect on GPIIb-IIIa expression, replacement with Glu, Lys, Pro, or Asn caused intracellular retention of GPIIb-IIIa. Concurrently, the consequences of these replacements were examined by comparative modeling by introducing them into the analogous position of the first helix-loop-helix (HLH) motif of calmodulin, based on homology between the calcium binding domains of GPIIb and the calcium binding loops of HLH-containing proteins. The modeling revealed that as the side chain of the introduced amino acid increased in size, it progressively interfered with hydrophobic interactions between the incoming and outgoing helices of the motif. To test whether this observation also applies to GPIIb, V286, located immediately distal to the first GPIIb calcium binding domain, was replaced by Asp and Phe. Expression of these mutants in COS-1 cells also resulted in the intracellular retention of GPIIb-IIIa, suggesting that interactions between sequences that flank the first calcium binding domain of GPIIb affect its folding. Finally, the endoplasmic reticulum chaperone BiP was detected in immunoprecipitates of GPIIb-IIIa containing GPIIb with Ala, Val, Lys, or Pro, but not Gly, at position 273. This suggests that although BiP binding is a sensitive indication of the fidelity of GPIIb-IIIa folding, it is not sufficient to account for the intracellular retention of the heterodimer.
Hematology-Oncology Division, Hospital of the University of Pennsylvania, Philadelphia, USA.
M J
MJ
Kahn
T
T
Kieber-Emmons
G
G
Vilaire
R
R
Murali
M
M
Poncz
J S
JS
Bennett
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000378:metabolism; D003198:Computer Simulation; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D046911:Macromolecular Substances; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D002118:Calcium