DISTINCT ROLES FOR ARGININES IN TRANSMEMBRANE HELIX-6 AND HELIX-7 OF THE THYROTROPIN-RELEASING-HORMONE RECEPTOR

The thyrotropin-releasing hormone (TRH) receptor (TRH-R) is a member o f the seven-transmembrane region, G protein-coupled receptor family. A rg-283 and Arg-306, in transmembrane helices 6 and 7, respectively, ar e putatively in positions homologous to those of residues that are imp ortant for agonist and antagonist binding in receptors for neurotransm itters. These arginines were mutated and the mutant receptors were tra nsiently expressed in COS-I cells. The affinity of the R306K TRH-R was similar to that of the wild-type (WT) TRH-R, whereas no specific bind ing was detected in cells expressing R306A, R306E, or R306L TRH-Rs. Be cause TRH stimulated inositol phosphate (IP) formation to similar maxi mal extents in cells expressing WT and Arg-306 mutant TRH-Rs, relative potencies were used to estimate the relative affinities of the recept ors. The EC,, values for stimulation of R306A, R306E, and R306L TRH-Rs were 1500-, 1200-, and 3000-fold higher than that for the WT TRH-R. N o specific binding was measurable in COS-1 cells expressing R283K, R28 3H, or R283A TRH-Rs, whereas maximal TRH stimulation of IP formation w as to levels 64%, 42%, or <1%, respectively, of that in cells expressi ng WT TRH-Rs; for R283K and R283H TRH-Rs, EC,, values were 6300- and 5 0,000-fold higher, respectively, than that for the WT TRH-R. In AtT-20 cells stably expressing R283A TRH-Rs, the binding affinity was 39,000 -fold lower than that of the WT TRH-R and the number of receptors was estimated to be 0.88 x 10(6)/cell, but TRH did not stimulate IP format ion. Thus, in the TRH-R, Arg-306 appears to be important for binding b ut not for activation, whereas Arg-283 appears to be important for bin ding and activation.