Total solid-phase synthesis and prolactin-inhibiting activity of the gonadotropin-releasing hormone precursor protein and the gonadotropin-releasing hormone associated peptide.

The human gonadotropin-releasing hormone precursor protein, pHGnRH (Met-23-Ile69) (preproGnRH), and three of its fragment peptides, pHGnRH (Asp14-Ile69) (gonadotropin-releasing hormone associated peptide--GAP), pHGnRH (Phe38-Ile69), and pHGnRH (Ser47-Ile69), were assembled in a stepwise solid-phase cosynthesis employing Boc/Bzl tactics and an optimized acylation schedule which included recoupling steps with hexafluoro-2-propanol to help overcome the aggregation of the pendant peptide chains of the peptidoresin during difficult couplings. Reversed-phase high-performance liquid chromatography (HPLC) purification yielded products which were characterized by analytical reversed-phase HPLC, ion-exchange chromatography, capillary zone electrophoresis, SDS-polyacrylamide gel electrophoresis, and ion-spray mass spectrometry to reveal a high degree of homogeneity. Biological characterization demonstrated that only GAP stimulated luteinizing hormone and follicle-stimulating hormone release from primary cultures of rat anterior pituitary cells, while GAP, pHGnRH (Phe38-Ile69), and preproGnRH all inhibited prolactin release, with the latter being the most potent at concentrations comparable to bromocryptine. However, only GAP and pHGnRH (Phe38-Ile69) were able to displace a labeled gonadotropin-releasing hormone agonist from binding to rat pituitary membrane preparations. This first demonstration of significant biological activity with a precursor protein also suggests that the gonadotropin-releasing and prolactin release-inhibiting functions of GAP are not mediated through the same pituitary receptors.