Human melanocortin receptor 2 expression and functionality: Effects of protein kinase A and protein kinase C on desensitization and internalization
Human melanocortin receptor 2 expression and functionality: Effects of protein kinase A and protein kinase C on desensitization and internalization
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DOI：
10.1210/en.2005-0991
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发表时间：
2006-05-01
期刊：
影响因子：
4.8
通讯作者：
Gallo-Payet, N
中科院分区：
文献类型：
--
作者：
Kilianova, Z;Basora, N;Gallo-Payet, N
本研究的目的是通过在M3细胞系中转染c-Myc标记的hMC 2 R来研究ACTH受体人（h）黑皮质素受体2（MC 2 R）的短期调节，并通过间接免疫荧光法评估其膜表达。促肾上腺皮质激素刺激诱导cAMP的产生，在瞬时和稳定转染子中的EC 50值分别为7.6-11.9 nM。用ACTH预处理诱导cAMP产生的剂量依赖性损失，从1 pM到10 nM。脱敏也是时间依赖性的，与70%的最大反应性的损失发生后15分钟的预处理与10 nM的ACTH，随后由一个平台高达60分钟。在hMC 2 R反应性的下降被废除的个别治疗与蛋白激酶A（PKA）或蛋白激酶C抑制剂，H-89和GF 109203 X。然而，当同时加入时，受体反应性高于对照细胞中观察到的最大hMC 2 R活性。ACTH诱导的cAMP产生的损失伴随着受体螯合到细胞内囊泡（最大后30分钟曝光）。用c-Myc标记的hMC 2 R和β-arrestin-2-绿色荧光蛋白与蔗糖处理一起共转染M3细胞，揭示了β-arrestin-2-绿色荧光蛋白和c-Myc-hMC 2 R通过网格蛋白依赖性但非小囊依赖性的过程在相似的细胞内囊泡中重新分布。蔗糖预处理阻断受体脱敏，表明hMC 2 R脱敏和内化相互关联。此外，与H-89预孵育废除hMC 2 R内化，而GF 109203 X没有影响。总之，本研究结果表明，PKA和蛋白激酶C协同作用，诱导hMC 2 R脱敏，但只有PKA是必不可少的受体内化，突出了这种受体的短期调控模式的复杂性。
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 EC50 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 companied 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.