IDENTIFICATION OF TRANSMEMBRANE REGIONS CRITICAL FOR LIGAND-BINDING TO THE HUMAN D-3 DOPAMINE-RECEPTOR USING VARIOUS D-3 D-1 TRANSMEMBRANE CHIMERAS/

To investigate the roles of individual transmembrane segments (TM) of the human D-3 dopamine receptor in its ligand-receptor interactions, w e generated chimeric receptors in which its TMs were replaced, one at a time, partially or entirely, by the corresponding TM of the homologo us human D-1 receptor. Ligand binding properties of the chimeras, as e xpressed heterologously in Sf9 cells using recombinant baculoviruses, indicate that the critical binding regions for D-3-selective (over D-1 ) ligands reside at narrow regions (6 to 8 residues) near the extracel lular surface for TMI, II, IV and VI, while TMV seems to be minimally involved in the ligand selectivity. For TMIII and TMVII, the critical regions seem to be deeper,involving at least the 10 residues near the extracellular surface for TMIII, and the entire TM segment for TMVII. This is based on our current observations that the chimeras with the, sequence in the critical regions, although the rest of the TM is of D- 1 origin (except TMVII), showed the binding properties indistinguishab le from those of the wild-type receptor. The chimeras with the D-1 seq uence in the regions, on the other hand, showed ligand binding charact eristics wildly variable depending on substituted TMs: Most marked dec reases in ligand affinities were observed with the chimeras of TMIII a nd VII, and intermediate changes with those of TMIV and VI. Replacemen ts of TMV produced no appreciable effects on the affinities of 14 test ligands (except for one). The chimeras of TMI and II with the D-1 seq uence in the critical regions showed no appreciable specific binding f or several radioactive D-3-selective ligands, possibly reflecting thei r critical roles in assembly and folding of the receptor. These critic al regions of the D-3 receptor were highly homologous to those of the D-2 receptor, except for several nonconservatively substituted residue s, which could be exploited to develop ligands selective for the D-3 o ver D-2 dopamine receptor or vice versa.