ANALYSIS OF STRUCTURAL REQUIREMENTS FOR TRH-POTENTIATING PEPTIDE RECEPTOR-BINDING BY ANALOG DESIGN

Previous studies established that the [I-125-Tyr(0)]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 [I-125-Tyr(0)]Ps4. The affinity of [Tyr(0)]Ps4 for rat anterior pituitary membranes [inhibitory constant (K-i), similar to 5 nM] was drastically reduced by the sustitution of either Tyr(0) with Gly or Asp(8) with Asn (K-i similar to 95 and similar to 51 nM, respectively). Deamination of [Tyr(0)]Ps4 also sharply reduced affinity (K-i similar to 1100 nM). In contrast, Ser(1) --> Ala, Pro3 --> Ala and Thr(10) --> Val substitutions led to analogues showing a tenfold increase in binding affinity relative to the parent peptide. The change of Phe(2) --> Leu, Trp(4) --> Ala, Glu(6) --> Gln, Val(9) --> Thr and carboxamidation of Thr(10) had no effect on binding affinity. The data suggest that substitutions of the amino-terminal group, Asp(8) and Tyr(0), have a marked effect on the ability of [Tyr(0)]Ps4 to compete with [I-125-Tyr(0)]Ps4 for binding to TRH-potentiating peptide pituitary receptor.