Chrm4

Muscarinic acetylcholine receptors (MAChRs, M receptors for short) are involved in motor control, thermoregulation, cardiovascular regulation and memory in the central nervous system, and mediate smooth muscle contraction, glandular secretion, and heart rate in the peripheral nervous system. Regulation of myocardial contractility. Therefore, studying the coupling mechanism between the subtypes of M receptors and their corresponding GTP binding proteins (G-proteins) is conducive to the development of drugs with strong M receptor selectivity to treat Alzheimer's disease, Parkinson's disease, asthma, analgesia, digestion, and dysfunction of internal organs such as the tract, heart, or bladder.

Muscarinic Acetylcholine Receptor Structure

Existing studies have shown that muscarinic acetylcholine receptors are a family of receptors that bind to G protein, and biological research divides them into five subtypes m1-m5. From a pharmacological point of view, the subtypes of M receptors are named M1, M2, M3, and M4, respectively. Because the protein expressed by the m5 gene has no corresponding functional pharmacological performance, there is no M5 for the time being. The molecular weight of the M receptor protein is about 51-66kD and consists of 460-590 amino acid residues. The N-terminus is outside the cell membrane and the C-terminus is inside the cytoplasm. It crosses the cell membrane seven times to form seven transmembrane hydrophobic regions (TM1- 7), Three outer membrane rings (O1-3) and three inner membrane rings (i1-3). The amino acid sequences of the five subtypes are very similar. Among them, the amino acid sequence of the TM1-7 transmembrane region is the closest. The O1-3 loop and the i1 and i2 loop sequences are also relatively conservative, but the N-terminal, C-terminal and i3 loops have greater variation. In particular, the i3 loop (157-240 amino acids), which has the longest amino acid sequence, has the largest difference, and is the main site where the M receptor binds to the G protein and causes biological effects. The seven transmembrane regions of the M receptor are arranged in a spoke shape on the cell membrane. Most of the conserved amino acid residues are located in the center, and the non-conserved residues face the lipids on the outside. This structure allows the M receptor to form a central, highly conserved hydrophilic depression that binds to the corresponding ligand.

Figure 1. The NGF interacts with CHRM4. (Wei-Yu Chen, et al.; 2021)

There are many amino acid residues in the M receptor molecule that are conserved in the superfamily of G protein-coupled receptors (GPCRs), which are essential to maintain the receptor structure and perform normal functions. The transmembrane region forms an alpha helix, in which TM 1 ~ 3 cross the cell membrane vertically, and TM 4-7 forms a 30° bend at the 2nd-4th helix proline outside the membrane; there is a conservative in O1 and O3 rings cysteine residues, the two form a disulfide bond to maintain a stable molecular conformation of the receptor protein; all M receptor ligands have a positively charged amino group, which is related to the aspartic acid at position 147 in TM3 The negatively charged carboxyl group forms an ionic bond, which is the way the ligand binds to the receptor; the tyrosine in TM 7 is very close to the aspartic acid mentioned above in space, and it also plays the role of ligand binding and receptor The effect of activation; there is a conserved triplet of amino acid residues (Asp-Arg-Ty r) at the junction of TM 3 and i2, which is essential for the expression and function of the receptor; there are one or more extracellular N-terminals There are several glycosylation sites, but glycosylation is not necessary for receptor expression and function; C-terminal palmitoylation occurs on cysteine residues near the cell membrane and participates in the coupling of G protein.

Muscarinic Acetylcholine Receptor Function

Generally speaking, M1, M3, and M5 subtypes are coupled with Gq/11 in G protein to activate the effector protein phospholipase C, which is called M1 group; while M2 and M4 are coupled with Gi or Go to inhibit Adenylate cyclase, called the M2 group. The two ends of the receptor i3 loop near the cell membrane continue the spiral structure of TM 5 and TM 6 to form a spiral, and they are adjacent to each other in space. The palmitic acid bound to the C-terminal cysteine is connected to the cell membrane at the other end to form the fourth intracellular loop (i4), which together with the helical structure of the i3 loop near the cell membrane forms the region that binds to the G protein, where i3 Tyr254 at the N-terminus of the ring is the key to determining the coupling between the two. The intracellular i1 and i2 loops also affect the recognition and coupling of the G protein. For example, the aspartic acid in the i2 loop of the M1 receptor is mutated to asparagine, although it can increase the affinity of carbachol to the receptor, but Reduces the ability of the receptor to couple with Gq to hydrolyze inositol phospholipids (PIP2). However, only a few amino acid residues in the i3 and i2 loops determine the specificity of the interaction between the M receptor and the G protein.

As an inhibitor of adenylate cyclase, CHRM4 involves a variety of cellular reactions, including inhibition of adenylate cyclase, decomposition of phosphoinositide, and regulation of potassium channels through the action of G protein. In addition, muscarinic receptors affect many functions of acetylcholine in the central and peripheral nervous system. The study shows that CHRM4 plays a key role in the progression of schizophrenia disease and expression of CHRM4 is reduced in hippocampus and caudal shell from schizophrenia patients. CHRM4 is related to the occurrence of asthma and the expression of CHRM4 is significantly increased in children with asthma. In addition, CHRM4 serves as a drug target and prognostic marker for schizophrenia. Studies have shown that M4 receptors can be used as drug targets for neurodegenerative diseases, and M4 receptor activators can be used as promising drugs for the treatment of central nervous system diseases.

References

1. Dohlman HG, et al.; Model systems for the study of seven-transmembrane-segment receptors . Annu Rev Biochem. 1991, 60: 653-688.
2. Hulme EC, et al.; Muscarinic receptor subtypes. Annu Rev Pharmacol Toxicol, 1990, 30: 633- 673 .
3. Kostenis E, et al.; Structure-function analysis of muscarinic receptors and their associated G proteins. Life Sci. 1999, 64 355-362.
4. Felder C C., et al.; Current status of muscarinic M1 and M4 receptors as drug targets for neurodegenerative diseases. Neuropharmacology. 2018, 136(Pt C): 449-458.
5. Takai K., et al.;Discovery and development of muscarinic acetylcholine M4 activators as promising therapeutic agents for CNS diseases. Chemical & Pharmaceutical Bulletin. 2018, 66(1): 37-44.
6. Joseph L., et al.; Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice. Behavioural Brain Research. 2017, 329: 75-83.
7. Wei-Yu Chen, et al.; Nerve growth factor interacts with CHRM4 and promotes neuroendocrine differentiation of prostate cancer and castration resistance.Communications Biology. 2021, 4: 22.