Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ibalpha.
Binding of the platelet GPIb/V/IX (glycoprotein Ib/V/IX) receptor to von Willebrand factor is critical for platelet adhesion and aggregation under conditions of rapid blood flow. The adhesive function of GPIbalpha is regulated by its anchorage to the membrane skeleton through a specific interaction with filamin A. In the present study, we examined the amino acid residues within the cytoplasmic tail of GPIbalpha, which are critical for association with filamin A, using a series of 25-mer synthetic peptides that mimic the cytoplasmic tail sequences of wild-type and mutant forms of GPIbalpha. Peptide binding studies of purified human filamin A have demonstrated a major role for the conserved hydrophobic stretch L567FLWV571 in mediating this interaction. Progressive alanine substitutions of triple, double and single amino acid residues within the Pro561-Arg572 region suggested an important role for Trp570 and Phe568 in promoting GPIbalpha binding to filamin A. The importance of these two residues in promoting filamin A binding to GPIbalpha in vivo was confirmed from the study of Chinese-hamster ovary cells expressing GPIbalpha Trp570-->Ala and Phe568-->Ala substitutions. Phenotypic analysis of these cell lines in flow-based adhesion studies revealed a critical role for these residues in maintaining receptor anchorage to the membrane skeleton and in maintaining cell adhesion to a von Willebrand factor matrix under high-shear conditions. These studies demonstrate a novel filamin A binding motif in the cytoplasmic tail of GPIbalpha, which is critically dependent on both Trp570 and Phe568.
Cranmer
Susan L
SL
Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Commercial Road, Prahran, Victoria 3181, Australia. sue.cranmer@med.monash.edu.au
Pikovski
Inna
I
Mangin
Pierre
P
Thompson
Philip E
PE
Domagala
Teresa
T
Frazzetto
Mark
M
Salem
Hatem H
HH
Jackson
Shaun P
SP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology; D003285:Contractile Proteins / Q000737:chemistry*; D006224:Cricetinae; D003593:Cytoplasm; D064448:Filamins; D015870:Gene Expression; D008840:Microfilament Proteins / Q000737:chemistry*; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010649:Phenylalanine; D014364:Tryptophan