Receptor-mediated internalization of LHRH antagonists by pituitary cells

A fluorescently labeled antagonist of luteinizing hormone releasing hormone (LHRH), d-pGlu-d-Phe-d-Trp-Ser-Tyr-d-Lys⁶-(tetramethylrhodamine)-Leu-Arg-Pro-Gly-NH₂, was prepared. This peptide retained high-affinity binding to the LHRH receptor of pituitary plasma membrane preparations. The analog was able to block LHRH-stimulated LH release from pituitaries incubated in vitro, and exhibited minor agonistic activity. This rhodamine-labeled antagonist was utilized for the microscopic visualization and localization of LHRH receptors in dispersed rat pituitary cells. The fluorescently labeled receptors were initially distributed uniformly on the cell surface. The hormone-receptor complexes were redistributed after incubation at 23 °C and formed clusters which subsequently became internalized (at 37°C) into endocytic vesicles. Addition of LHRH (10^-6 M) abolished these processes, indicating specific binding sites for the rhodamine-labeled peptide to the gonadotrope cells, A quantitative comparison of temperature-dependent internalization by iodinated LHRH agonist and antagonist revealed that both analogs were internalized to a similar extent. These findings suggest that LHRH-receptor complex internalization is related to LHRH receptor regulation.