h-89 and sauvagine

The corticotropin releasing factor receptor 1 (CRFR1) belongs to the superfamily of G-protein coupled receptors. Though CRF is involved in the aetiology of several stress-related disorders, including depression and anxiety, details of CRFR1 regulation such as internalization remain uncharacterized. In the present study, agonist-induced internalization of CRFR1 in HEK293 cells was visualized by confocal microscopy and quantified using the radioligand 125I-labelled sauvagine. Recruitment of beta-arrestin 1 in response to receptor activation was demonstrated by confocal microscopy. The extent of 125I-labelled sauvagine stimulated internalization was significantly impaired by sucrose, indicating the involvement of clathrin-coated pits. No effect on the extent of internalization was observed in the presence of the second messenger dependent kinase inhibitors H-89 and staurosporine, indicating that cAMP-dependent protein kinase and protein kinase C are not prerequisites for CRFR1 internalization. Surprisingly, deletion of all putative phosphorylation sites in the C-terminal tail, as well as a cluster of putative phosphorylation sites in the third intracellular loop, did not affect receptor internalization. However, these mutations almost abolished the recruitment of beta-arrestin 1 following receptor activation. In conclusion, we demonstrate that CRFR1 internalization is independent of phosphorylation sites in the C-terminal tail and third intracellular loop, and the degree of beta-arrestin 1 recruitment.

Topics: Amphibian Proteins; Animals; Arrestins; beta-Arrestin 1; beta-Arrestins; Binding Sites; Cattle; Cell Line; Clathrin-Coated Vesicles; Cyclic AMP-Dependent Protein Kinases; Endocytosis; Humans; Immunohistochemistry; Isoquinolines; Microscopy, Confocal; Mutagenesis, Site-Directed; Peptide Hormones; Peptides; Phosphorylation; Protein Kinase C; Radioligand Assay; Receptors, Corticotropin-Releasing Hormone; Recombinant Proteins; Staurosporine; Sulfonamides

Intracellular Ca2+ oscillations play an important role in the induction of alpha-MSH release from pituitary melanotrope cells of Xenopus laevis. Oscillatory, secretory and adenylyl cyclase activities are all inhibited by dopamine, neuropeptide Y (NPY) and baclofen (a GABAB receptor agonist) and stimulated by sauvagine. In this study, we test the hypothesis that these neural messengers regulate the Ca2+ oscillations via a cAMP/protein kinase A (PKA)-dependent mechanism. To this end, video-imaging microscopy was applied to single Xenopus melanotropes loaded with the Ca2+ indicator Fura-2. The cAMP-dependent PKA inhibitor H89 blocked Ca2+ oscillations as well as the stimulatory actions of 8-Br-cAMP and sauvagine. Treatment of cells inhibited by baclofen with either 8-Br-cAMP or sauvagine led to a reappearance of Ca2+ oscillations. A similar result was found for cells inhibited by NPY. Neither 8-Br-cAMP nor sauvagine induced Ca2+ oscillations in cells inhibited by dopamine. Depolarizing dopamine-inhibited cells with high potassium also failed to induce oscillations, but combining 8-Br-cAMP with membrane depolarization induced oscillations. It is concluded that sauvagine, baclofen and NPY work primarily through a cAMP/PKA-pathway while dopamine inhibits Ca2+ oscillations in a dual fashion, namely via both a cAMP-dependent and a cAMP-independent mechanism, the latter probably involving membrane hyperpolarization.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amphibian Proteins; Animals; Baclofen; Calcium; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dopamine; Drug Interactions; Enzyme Inhibitors; Female; Fluorescent Dyes; Fura-2; GABA Agonists; Isoquinolines; Male; Microscopy, Video; Neuropeptide Y; Peptide Hormones; Peptides; Periodicity; Pituitary Gland; Sulfonamides; Vasodilator Agents; Xenopus laevis