STERIC HINDRANCE MUTAGENESIS VERSUS ALANINE SCAN IN MAPPING OF LIGAND-BINDING SITES IN THE TACHYKININ NK1 RECEPTOR

Residues in transmembrane domain (TM)-III, TM-V, TM-VI, and TM-VII bel ieved to be facing the deep part of the presumed main ligand-binding p ocket of the NK1, receptor were probed by alanine substitution and int roduction of residues with larger and/or chemically distinct side chai ns. Unaltered or even improved binding affinity for four peptide agoni sts, substance P, substance P-O-methyl ester, eledoisin, and neurokini n A, as well as normal EC50 values for substance P in stimulating phos phatidylinositol turnover indicated that these mutations did not alter the overall functional integrity of the receptor. The alanine substit utions in general had only minor effects on nonpeptide antagonist bind ing. However, the introduction of the larger and polar aspartic acid a nd histidine residues at positions corresponding to the monoamine bind ing aspartic acid in TM-III of the beta(2)-adrenoceptor (ProIII:08, Pr o112 in the NK1, receptor) and to the presumed monoamine interacting ' 'two serines'' in TM-V (ThrV:09, Thr201; and IIeV:12, IIe204) impaired by >100-fold the binding of a group of nonpeptide antagonists, includ ing CP96,345, CP99,994, RP67,580, RPR100,893, and CAM4092. In contrast , another group of nonpeptide antagonists, LY303,870, FK888, and SR140 ,333, were little or not at all affected by the space-filling substitu tions. Two of these compounds, FK888 and LY303,870, were those most se riously affected (75-89-fold) by alanine substitution of PheVI:20 loca ted in the upper part of the main ligand-binding crevice. Surprisingly , substitution of AlaIII:ll (Ala115), which is located in the middle o f TM-III, conceivably pointing toward TM-VII, with a larger valine res idue increased the affinity for all 13 ligands tested, presumably by c reating a closer interhelical packing. It is concluded that the introd uction of larger side chains at positions at which molecular models in dicate that this is structurally allowed can be a powerful method of l ocating ligand-binding sites due to the considerable difference betwee n positive and negative results. Such steric hindrance mutagenesis str ongly indicates that one population of nonpeptide antagonists bind in the deep pocket of the main ligand-binding crevice of the NK1, recepto r, whereas another group of nonpeptide antagonists, especially SR140,3 33, was surprisingly resistant to mutational mapping in this pocket.