title: Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity.
abstract: When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor1. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins.
authors.affiliation: Department of Biological Sciences, Lehigh University.
authors.forename: Sara Lynn N
authors.initials: SL
authors.lastname: Farwell
authors.affiliation: Department of Natural Science, DeSales University.
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Slee
authors.affiliation: Department of Biological Sciences, Lehigh University.
authors.forename: Yaqiu
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, Lehigh University; ljl0@lehigh.edu.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Lowe-Krentz
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008565:Membrane Proteins; D011956:Receptors, Cell Surface / Q000378:metabolism* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D033921:Vesicular Transport Proteins
chemical_list: D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C000609639:Tmem184a protein, human; D033921:Vesicular Transport Proteins; C037680:heparin receptor; D049452:Green Fluorescent Proteins