STRUCTURAL DETERMINANTS FOR AGONIST BINDING-AFFINITY TO THROMBOXANE PROSTAGLANDIN ENDOPEROXIDE (TP) RECEPTORS - ANALYSIS OF CHIMERIC RAT/HUMAN TP RECEPTORS/

The two most extensively characterized thromboxane/prostaglandin endop eroxide (TP) receptors, from human platelets and rat vascular smooth m uscle, exhibit thromboxane agonist [15-(1 alpha,2 beta(5Z),3 alpha-(1E ,3S),4 -4-(p-iodophenoxy)-1-butenyl-7-oxabicycloheptenoic acid (I-BOP) binding affinities that differ by an order of magnitude, rat TP havin g the higher affinity, We utilized this difference in I-BOP affinity t o identify structural determinants of TP receptor heterogeneity, No si gnificant difference was found in the rank order of affinities for a s eries of thromboxane receptor ligands to bind to cloned human TP alpha versus rat TP, indicating that these represent species homologs, not distinct TP subtypes, Structural determinants for observed differences in I-BOP binding K-d 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 transmembran es 1, 2, 4, or 7 were constructed and expressed in HEK293 cells for an alysis of ligand binding properties, Substitution of any part except t he carboxyl tail of the human TP into the rat TP resulted in a recepto r with I-BOP binding affinity intermediate between the two, Analysis o f chimeras in which only the extracellular amino terminus and a portio n of transmembrane 1 were switched localized the determinant of high a ffinity binding to the region between amino acids 3 and 40, Using this chimera, amino acids in the human portion (extracellular amino termin us and part of transmembrane 1) were replaced with analogous amino aci ds from rat TP to regain high affinity I-BOP binding, Only when amino acid Val(37) and either Val(36) or Ala(40) were reverted to their resp ective rat TP counterparts (Ala(36), Leu(37), and Gly(40), respectivel y) was high affinity I-BOP binding recovered, The mechanism for the in creased I-BOP affinity may be the lengthening of the amino acid side c hain at position 37, thus extending this group further into the putati ve I-BOP binding pocket, with compensatory shortening of side chains i n spatially adjacent amino acids.