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.
Frändberg. P A PA; Doufexis. M M; Kapas. S S; Chhajlani. V V
Key Findings
- Reducing disulfide bonds with DTT weakens alpha‑MSH binding but not NDP‑MSH binding.
- Cysteines in extracellular loops are needed for ligand binding; cysteines in transmembrane and intracellular regions are needed for signaling.
- A single C78G mutation lets alpha‑MSH work normally but turns NDP‑MSH into an antagonist.
Practical Outcomes
- For biohackers using melanotan‑I, the main takeaway is that individual genetic variations in the MC1R receptor may affect how well the peptide works. This study doesn’t change dosing or protocol, but suggests that people with certain MC1R mutations might experience reduced or altered effects, so personal response could vary.
Summary
This study shows that specific cysteine parts of the skin‑color receptor (MC1R) are crucial for how it binds and reacts to hormone‑like peptides such as alpha‑MSH (the natural version) and synthetic versions like NDP‑MSH. Changing one cysteine (C78) makes the synthetic peptide stop working as an activator and instead block the receptor. While the work is very detailed and done in cells, it hints that natural genetic differences in MC1R could change how people respond to melanotan‑I and similar compounds.
Abstract
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.
Study Information
pubmed
2001
2001-03-09T00:00:00.000Z
10.1006/bbrc.2001.4429