Zhang Jiannan, Li Xin, Zhou Yawei, Cui Lin, Li Jing, Wu Chenlei, Wan Yiping, Li Juan, Wang Yajun
Key Laboratory of Bio-resources and Eco-environment of Ministry of EducationCollege of Life Sciences, Sichuan University, Chengdu, People's Republic of China.
Key Laboratory of Bio-resources and Eco-environment of Ministry of EducationCollege of Life Sciences, Sichuan University, Chengdu, People's Republic of China
J Endocrinol. 2017 Aug;234(2):155-174. doi: 10.1530/JOE-17-0131. Epub 2017 May 16.
The interaction of melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors with proopiomelanocortin (POMC)-derived peptides (e.g. α-MSH), agouti-related protein (AgRP) and melanocortin-2 receptor accessory protein 2 (MRAP2) is suggested to play critical roles in energy balance of vertebrates. However, evidence on their interaction in birds remains scarce. Our study aims to reveal their interaction in chickens and the results showed that (1) chicken (c-)MC3R and cMC4R expressed in Chinese hamster ovary (CHO) cells can be activated by α-MSH and ACTH equipotently, monitored by a pGL3-CRE-luciferase reporter system; (2) cMC3R and cMC4R, when co-expressed with cMRAP2 (or cMRAP, a cMRAP2 homolog), show increased sensitivity to ACTH treatment and thus likely act as ACTH-preferring receptors, and the interaction between cMC3R/cMC4R and cMRAP2 was demonstrated by co-immunoprecipitation assay; (3) both cMC3R and cMC4R display constitutive activity when expressed in CHO cells, as monitored by dual-luciferase reporter assay, and cMRAP2 (and cMRAP) can modulate their constitutive activity; (4) AgRP inhibits the constitutive activity of cMC3R/cMC4R, and it also antagonizes ACTH/α-MSH action on cMC4R/cMC3R, indicating that AgRP functions as the inverse agonist and antagonist for both receptors. These findings, together with the co-expression of , , , and in chicken hypothalamus detected by quantitative real-time PCR, suggest that within the hypothalamus, α-MSH/ACTH, AgRP and MRAP2 may interact at the MC4R(/MC3R) interface to control energy balance. Furthermore, our data provide novel proof for the involvement of MRAP2 (and MRAP) in fine-tuning the constitutive activity and ligand sensitivity and selectivity of both MC3R and MC4R in vertebrates.
黑素皮质素 - 4（MC4R）和黑素皮质素 - 3（MC3R）受体与阿黑皮素原（POMC）衍生肽（如α - MSH）、刺鼠相关蛋白（AgRP）以及黑素皮质素 - 2受体辅助蛋白2（MRAP2）之间的相互作用，被认为在脊椎动物的能量平衡中起着关键作用。然而，关于它们在鸟类中相互作用的证据仍然很少。我们的研究旨在揭示它们在鸡中的相互作用，结果表明：（1）通过pGL3 - CRE - 荧光素酶报告系统监测，在中国仓鼠卵巢（CHO）细胞中表达的鸡（c - ）MC3R和cMC4R可被α - MSH和促肾上腺皮质激素（ACTH）同等程度地激活；（2）当cMC3R和cMC4R与cMRAP2（或cMRAP，一种cMRAP2同源物）共表达时，对ACTH处理表现出更高的敏感性，因此可能作为更倾向于ACTH的受体，并且通过共免疫沉淀试验证明了cMC3R/cMC4R与cMRAP2之间的相互作用；（3）通过双荧光素酶报告试验监测，当在CHO细胞中表达时，cMC3R和cMC4R均表现出组成性活性，并且cMRAP2（和cMRAP）可以调节它们的组成性活性；（4）AgRP抑制cMC3R/cMC4R的组成性活性，并且它还拮抗ACTH/α - MSH对cMC4R/cMC3R的作用，表明AgRP作为这两种受体的反向激动剂和拮抗剂发挥作用。这些发现，连同通过定量实时PCR检测到的鸡下丘脑、、、和的共表达，表明在下丘脑中，α - MSH/ACTH、AgRP和MRAP2可能在MC4R（/MC3R）界面相互作用以控制能量平衡。此外，我们的数据为MRAP2（和MRAP）参与微调脊椎动物中MC3R和MC4R的组成性活性以及配体敏感性和选择性提供了新的证据。
