Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif.
The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets.
Centre for Sythesis and Chemical Biology, Department of Chemistry, Royal College of Surgeons in Ireland, Dublin.
Dermot
D
Reilly
Deirdre
D
Larkin
Marc
M
Devocelle
Desmond J
DJ
Fitzgerald
Niamh
N
Moran
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry; D019921:Biotinylation; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D037282:Calreticulin / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D002449:Cell Aggregation / Q000502:physiology; D003432:Cross-Linking Reagents / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D005340:Fibrinogen / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D040201:Platelet Membrane Glycoprotein IIb / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D011107:Polylysine / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000032:analysis / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry; D016608:Talin / Q000032:analysis / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D037282:Calreticulin; D003432:Cross-Linking Reagents; D003902:Detergents; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D040201:Platelet Membrane Glycoprotein IIb; D021122:Protein Subunits; D013388:Succinimides; D016608:Talin; C093483:neutravidin; D001360:Avidin; D011107:Polylysine; D005340:Fibrinogen; D004492:Edetic Acid; D013917:Thrombin; D019704:Protein Disulfide-Isomerases; C011240:dithiobis(succinimidylpropionate); D002118:Calcium