Frändberg P A, Doufexis M, Kapas S, Chhajlani V
Division of Biological Research on Drug Dependence, Biomedical Centre, Uppsala, S-751 24, Sweden.
Biochem Biophys Res Commun. 2001 Mar 9;281(4):851-7. doi: 10.1006/bbrc.2001.4429.
Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.
随着二硫苏糖醇（DTT）浓度增加，人促黑素细胞激素1受体（hMC1R）中的二硫键减少，导致[125I]-促肾上腺皮质激素（ACTH，L-异构体）的结合呈单相下降，而[125I]-NDP-MSH（[Nle(4),D-Phe(7)]-α-黑素细胞刺激素；D-异构体）的结合呈双相下降。用10 mM DTT预处理hMC1R导致对α-MSH（L-异构体）的亲和力丧失36倍，而不影响NDP-MSH（D-异构体）的亲和力。为了确定各个半胱氨酸残基的作用，我们采用定点诱变将hMC1R中所有14个位置的半胱氨酸替换为甘氨酸，并分析野生型和突变型受体的配体结合和cAMP信号传导。细胞外环中四个半胱氨酸残基单点突变为甘氨酸（C35G、C267G、C273G和C275G）导致[125I]-NDP-MSH的结合完全丧失。此外，在C191G（跨膜段5）、C215G（第三细胞内环）和C315G（C末端环）位置具有正常配体结合的突变体，对激动剂α-MSH和NDP-MSH均未能产生cAMP信号。C78G位置的突变体（对α-MSH和NDP-MSH具有野生型结合）对α-MSH产生cAMP信号（与野生型hMC1R相同），但有趣的是不能被NDP-MSH刺激。此外，这种单氨基酸取代使NDP-MSH在C78G突变体受体上从激动剂转变为拮抗剂。这些发现表明：（i）α-MSH和ACTH（L-异构体）与D-异构体NDP-MSH在受体结合对DTT的敏感性方面不同；（ii）N端和细胞外环3中的半胱氨酸残基形成二硫键，是hMC1R结构完整性所必需的；（iii）跨膜段和细胞内环中的半胱氨酸残基是受体-G蛋白偶联所必需的；（iv）跨膜段2中的C78是D-异构体激动剂（NDP-MSH）产生功能性反应所必需的，但不是L-异构体激动剂（α-MSH）所必需的；（v）野生型受体激动剂NDP-MSH在突变体C78G受体上是拮抗剂。
