title: Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain.
abstract: Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.
authors.affiliation: Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: William T
authors.initials: WT
authors.lastname: Barry
authors.forename: Christel
authors.initials: C
authors.lastname: Boudignon-Proudhon
authors.forename: David D
authors.initials: DD
authors.lastname: Shock
authors.forename: Andrew
authors.initials: A
authors.lastname: McFadden
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Weiss
authors.forename: John
authors.initials: J
authors.lastname: Sondek
authors.forename: Leslie V
authors.initials: LV
authors.lastname: Parise
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D019703:Calcineurin / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins; D051597:Hippocalcin; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins; D010455:Peptides / Q000737:chemistry; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D051603:Recoverin; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C496731:RCVRN protein, human; D011994:Recombinant Proteins; D051603:Recoverin; D051597:Hippocalcin; D014364:Tryptophan; D019703:Calcineurin