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  • Title: Structural determinants for agonist binding affinity to thromboxane/prostaglandin endoperoxide (TP) receptors. Analysis of chimeric rat/human TP receptors.
    Author: Dorn GW, Davis MG, D'Angelo DD.
    Journal: J Biol Chem; 1997 May 09; 272(19):12399-405. PubMed ID: 9139686.
    Abstract:
    The two most extensively characterized thromboxane/prostaglandin endoperoxide (TP) receptors, from human platelets and rat vascular smooth muscle, exhibit thromboxane agonist [15-(1alpha,2beta(5Z), 3alpha-(1E,3S), 4alpha)]-7-[3-hydroxy-4-(p-iodophenoxy)-1-butenyl-7-oxabi cyclohepteno ic acid (I-BOP) binding affinities that differ by an order of magnitude, rat TP having the higher affinity. We utilized this difference in I-BOP affinity to identify structural determinants of TP receptor heterogeneity. No significant difference was found in the rank order of affinities for a series of thromboxane receptor ligands to bind to cloned human TPalpha versus rat TP, indicating that these represent species homologs, not distinct TP subtypes. Structural determinants for observed differences in I-BOP binding Kd were localized by creating chimeric human/rat TP followed by mutational substitution of specific critical amino acids. Initially, seven chimeric receptors with splice sites in transmembranes 1, 2, 4, or 7 were constructed and expressed in HEK293 cells for analysis of ligand binding properties. Substitution of any part except the carboxyl tail of the human TP into the rat TP resulted in a receptor with I-BOP binding affinity intermediate between the two. Analysis of chimeras in which only the extracellular amino terminus and a portion of transmembrane 1 were switched localized the determinant of high affinity binding to the region between amino acids 3 and 40. Using this chimera, amino acids in the human portion (extracellular amino terminus and part of transmembrane 1) were replaced with analogous amino acids from rat TP to regain high affinity I-BOP binding. Only when amino acid Val37 and either Val36 or Ala40 were reverted to their respective rat TP counterparts (Ala36, Leu37, and Gly40, respectively) was high affinity I-BOP binding recovered. The mechanism for the increased I-BOP affinity may be the lengthening of the amino acid side chain at position 37, thus extending this group further into the putative I-BOP binding pocket, with compensatory shortening of side chains in spatially adjacent amino acids.
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