ABSTRACT
Binding of a coagulation factor VIII to phosphatidylserine-containing membranes is critical for exerting its cofactor activity. The use of surface plasmon resonance allows studying factor VIII interaction with immobilized phospholipids. In the present study we compared factor VIII-binding properties of phospholipid surfaces immobilized on L1 and HPA Biacore chips in the form of a flexible bilayer and rigid monolayer, respectively. We demonstrated that immobilized phospholipid surfaces with physiological contents of PS and PE formed on L1 but not on HPA chip closely mimic intact phospholipid vesicles in their factor VIII and thrombin-activated factor VIII (factor VIIIa) binding properties.
Subject(s)
Biosensing Techniques , Factor VIII/metabolism , Phospholipids/chemistry , Protein Binding , Surface PropertiesABSTRACT
The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D(3) receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Carrier Proteins/genetics , Dimerization , Furylfuramide/metabolism , Mediator Complex Subunit 1 , Mice , Molecular Sequence Data , Mutagenesis , Receptors, Calcitriol/metabolism , Receptors, Retinoic Acid/metabolism , Receptors, Thyroid Hormone/metabolism , Retinoid X Receptors , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.
