Cysteine residues are involved in structure and function of melanocortin 1 receptor:: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist
Cysteine residues are involved in structure and function of melanocortin 1 receptor:: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist
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DOI：
10.1006/bbrc.2001.4429
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发表时间：
2001-03-09
影响因子：
3.1
通讯作者：
Chhajlani, V
中科院分区：
文献类型：
--
作者：
Frändberg, PA;Doufexis, M;Chhajlani, V
随着DTT（二硫苏糖醇）浓度的增加，人黑皮质素1受体（hMC 1 R）中二硫键的还原导致[I-125]-ACTH（促肾上腺皮质激素，L-异构体）结合以单相方式降低，[I-125]-NDP-MSH（[Nle（4），D-Phe（7）]-α-黑素细胞刺激激素; D-异构体）结合以双相方式降低。用10 mM DTT预处理hMC 1 R导致对α-MSH（L-异构体）的亲和力损失36倍，而不影响NDP-MSH（D-异构体）的亲和力。为了表征单个半胱氨酸残基的作用，我们采用定点突变在hMC 1 R中的所有14个位置用甘氨酸取代半胱氨酸，并分析了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4个半胱氨酸残基的单点突变为甘氨酸（C35 G、C267 G、C273 G和C275 G）导致[I-125]-NDP-MSH结合完全丧失。此外，在位置C191 G（跨膜区段5）、C215 G（第三胞内环）和C315 G（C-末端环）处具有正常配体结合的突变体不能响应于激动剂α-MSH和NDP-MSH产生cAMP信号。在位置C78 G处的突变体（具有与α-MSH以及NDP-MSH的野生型结合）响应于α-MSH（与野生型hMC 1 R相同）产生cAMP信号，但有趣的是不能被NDP-MSH刺激。此外，该单个氨基酸取代将NDP-MSH从C78 G突变体受体的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的不同之处在于它们对受体结合的DTT的敏感性，（ii）N-末端和细胞外环3中的半胱氨酸残基形成二硫键，并且是hMC 1 R的结构完整性所需的，（iii）跨膜区段和细胞内环中的半胱氨酸残基是受体-G-蛋白偶联所需的，（iv）跨膜区段2中的C78是D-异构体激动剂（NDP-MSH）而不是L-异构体激动剂（α-MSH）产生功能应答所需的，和（v）野生型受体激动剂NDP-MSH是突变C78 G受体的拮抗剂。(C)北京：科学出版社.
Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentration of DTT (dithiothreitol) resulted in a decrease in the binding of [I-125]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [I-125]-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 [I-125]-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 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 mutant C78G receptor. (C) 2001 Academic Press.