Analysis of structural requirements for TRH-potentiating peptide receptor binding by analogue design

Previous studies established that the [125I-Tyr0]Ps4 derivative of TRH-potentiating peptide (Ps4), Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr, displays high affinity and selectivity for an orphan membrane receptor in rat anterior pituitary. To identify the sites in Ps4 that determine receptor binding affinity, we have synthesized and screened a panel of 15 single-point substituted analogues for their ability to displace bound [125I-Tyr0]Ps4. The affinity of [Tyr0]Ps4 for rat anterior pituitary membranes [inhibitory constant (Ki), approximately 5 nM] was drastically reduced by the substitution of either Tyr0 with Gly or Asp8 with Asn (Ki approximately 95 and approximately 51 nM, respectively). Deamination of [Tyr0]Ps4 also sharply reduced affinity (Ki approximately 1100 nM). In contrast, Ser1-->Ala, Pro3-->Ala and Thr10-->Val substitutions led to analogues showing a tenfold increase in binding affinity relative to the parent peptide. The change of Phe2-->Leu, Trp4-->Ala, Glu6-->Gln, Val9-->Thr and carboxamidation of Thr10 had no effect on binding affinity. The data suggest that substitutions of the amino-terminal group, Asp8 and Tyr0, have a marked effect on the ability of [Tyr0]Ps4 to compete with [125I-Tyr0]Ps4 for binding to TRH-potentiating peptide pituitary receptor.