Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2011;6(9):e24644. doi: 10.1371/journal.pone.0024644. Epub 2011 Sep 13.
The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturally-occurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been identified in melanic mice and several other species. This mutation induces a pronounced increase in MC1R constitutive activity suggesting a link between constitutive activity and melanism which is corroborated by the attenuation of α-melanocyte stimulating hormone (αMSH) induced activation. However, the mechanism by which the mutation induces constitutive activity is currently not known.
METHODOLOGY/PRINCIPAL FINDINGS: Here we characterize the constitutive activity, cell surface expression and internalization of the mouse mutant, Mc1r E92K. As previously reported, only positively charged residues at position II:20/2.60 induced an increase in constitutive activity as measured by cAMP accumulation and CREB activation. Furthermore, the mutation induced a constitutive recruitment of β-arrestin. This phenomenon is only observed in MC1R, however, as the equivalent mutations in MC2-5R had no effect on receptor signaling. Interestingly, the mutation did not induce constitutive ERK1/2 phosphorylation or increase the internalization rate indicating the constitutive activity to be biased. Finally, to identify regions of importance for the increased constitutive activity of Mc1r E92K, we employed a chimeric approach and identified G102 and L110 in the extracellular loop 1 to be selectively important for the constitutive activity as this, but not αMSH-mediated activation, was abolished upon Ala substitution.
CONCLUSIONS/SIGNIFICANCE: It is concluded that the E92K mutation induces an active conformation distinct from that induced by αMSH and that the extracellular loop 1 is involved in maintaining this conformational state. In turn, the results suggest that in MC1R, which lacks an extracellular loop 2, the first extracellular loop may play a more prominent role during receptor activation than in general.
黑素皮质素 1 受体 (MC1R) 是黑色素形成的关键调节剂。因此，许多自然发生的 MC1R 突变与颜色变化有关。一个例子是在跨膜螺旋 II 的顶部位置 II:20/2.60 发现的谷氨酸到赖氨酸取代，该取代已在黑色素小鼠和其他几种物种中被发现。这种突变诱导 MC1R 组成型活性的显著增加，表明组成型活性与黑色素形成之间存在联系，这一联系得到 α-促黑素细胞激素 (αMSH) 诱导的激活减弱的证实。然而，目前尚不清楚突变如何诱导组成型活性。
方法/主要发现：在这里，我们对小鼠突变体 Mc1r E92K 的组成型活性、细胞表面表达和内化进行了表征。如前所述，只有位置 II:20/2.60 上的正电荷残基才能诱导组成型活性的增加，这可以通过 cAMP 积累和 CREB 激活来衡量。此外，该突变诱导β-arrestin 的组成型募集。这种现象仅在 MC1R 中观察到，然而，在 MC2-5R 中的等效突变对受体信号没有影响。有趣的是，该突变不会诱导组成型 ERK1/2 磷酸化或增加内化率，表明组成型活性具有偏向性。最后，为了确定 Mc1r E92K 组成型活性增加的重要区域，我们采用嵌合方法，发现细胞外环 1 中的 G102 和 L110 对组成型活性特别重要，因为这种活性，但不是 αMSH 介导的激活，在 Ala 取代后被消除。
结论/意义：可以得出结论，E92K 突变诱导了一种不同于 αMSH 诱导的活性构象，并且细胞外环 1 参与维持这种构象状态。反过来，结果表明，在缺乏细胞外环 2 的 MC1R 中，第一个细胞外环在受体激活过程中可能比一般情况下发挥更突出的作用。
