Cytochemical studies of corticotropin-releasing factor (CRF) receptors in anterior lobe corticotropes: binding, glucocorticoid regulation, and endocytosis of [biotinyl-Ser1]CRF.

A new synthetic biotinylated N-terminal analog of CRF was used to study its binding, endocytosis, and route of processing. The new analog was fully bioactive compared to rat(r) CRF in an ACTH bioassay and pituitary membrane receptor assay. In 1- to 4-day cultured pituitary cell monolayers, CRF receptors were labeled for 1-biotinylated rCRF (1-bio-CRF) and demonstrated with avidin-fluorescein (Ar-Fl) in living cells or with avidin-biotin-peroxidase complex (ABC) in fixed cells. The percentages of fluorescein-labeled cells were comparable to those stained with the ABC technique. The specificity of binding by 1-bio-CRF was shown by the ability of unlabeled CRF to inhibit staining, whereas 100 nM arginine vasopressin, angiotensin II, or somatostatin was without effect. Treatment of the cells with 100 nM glucocorticoids for 1 or 24 h before the 3-min stimulation with 1-bio-CRF caused a 50-60% reduction in the percentage of cells stained with either ABC or Av-Fl. Pretreatment with vehicle, a lower dose of corticosterone (10 nM), or other steroids (dihydrotestosterone or epitestosterone) did not decrease the percentage of cells stained with the ABC technique. The lower dose of glucocorticoids decreased the stain with Av-Fl by 50% after 1 h of pretreatment. Electron microscopic analysis of binding and endocytosis of 1-bio-CRF stained with the ABC technique showed patches of stain on coated pits and microvilli during the first 3 min of exposure. Internalization in vesicles and receptosomes was also seen during the first 3 min. Some stain was found on secretion granules as early as 1 min after exposure. Five minutes after exposure, the stain was in receptosomes, Golgi cisternae, condensing vesicles in the transreticular Golgi region, and 23% of the granules in the cytoplasm. During the later exposure periods (15-30 min), stain was also seen in multivesicular bodies. The correlative light and electron microscopic studies showed that 1-bio-CRF is rapidly internalized after patching on the surface and is ultimately found in multivesicular bodies or granules. We postulate that these structures are involved in processing or degradation of the ligand.