Human melanocortin receptor 2 expression and functionality: effects of protein kinase A and protein kinase C on desensitization and internalization.
The aim of this study was to investigate the short-term regulation of the ACTH receptor human (h) melanocortin receptor 2 (MC2R) by transfection of a c-Myc-tagged hMC2R in the M3 cell line and assess its membrane expression by indirect immunofluorescence. Stimulation with ACTH induced production of cAMP with EC(50) values ranging from 7.6-11.9 nM in transient and stable transfectants, respectively. Pretreatment with ACTH induced a dose-dependent loss of cAMP production, from 1 pm up to 10 nM. Desensitization was also time dependent, with 70% loss of maximal responsiveness occurring after 15-min pretreatment with 10 nM ACTH, followed by a plateau up to 60 min. The decrease in hMC2R responsiveness was abrogated by individual treatment with protein kinase A (PKA) or protein kinase C inhibitors, H-89 and GF109203X. However, when added simultaneously, receptor responsiveness was raised over the maximal hMC2R activity observed in control cells. ACTH-induced loss of cAMP production was accompanied by receptor sequestration into intracellular vesicles (maximum after 30-min exposure). Cotransfection of M3 cells with the c-Myc-tagged hMC2R and beta-arrestin-2-green fluorescence protein along with sucrose treatment revealed that beta-arrestin-2-green fluorescence protein and c-Myc-hMC2R were redistributed in similar intracellular vesicles through a clathrin-dependent, but caveolae-independent, process. Sucrose pretreatment blocked receptor desensitization, indicating that hMC2R desensitization and internalization are interrelated. Moreover, preincubation with H-89 abrogated hMC2R internalization, whereas GF109203X had no effect. In conclusion, the present results indicate that PKA and protein kinase C act synergistically to induce hMC2R desensitization, but only PKA is essential for receptor internalization, highlighting the complex nature of the short-term regulatory pattern of this receptor.
Kilianova
Zuzana
Z
Service d'Endocrinologie, Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Québec, Canada.
Basora
Nuria
N
Kilian
Peter
P
Payet
Marcel D
MD
Gallo-Payet
Nicole
N
D019390:Arrestins / Q000096:biosynthesis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D044103:Receptor, Melanocortin, Type 2 / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D004791:Enzyme Inhibitors; D000939:Epitopes; D007211:Indoles; D008301:Maleimides; D044103:Receptor, Melanocortin, Type 2; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D013395:Sucrose; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C070515:bisindolylmaleimide I