The Role of the DRY Motif of Human MC4R for Receptor Activation

Yamano, Yoshiaki; Kamon, Rio; Yoshimizu, Takao; Toda, Yoshihisa; Oshida, Yoshiki; Chaki, Shigeyuki; Yoshioka, Masanobu; Morishima, Isao

doi:10.1271/bbb.68.1369

Cited by 11 publications

(11 citation statements)

References 15 publications

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“…1, excluding previously well-studied S136 (Fan & Tao 2009) and DRYFTI motifs (Tao & Segaloff 2004, Yamano et al 2004, Govaerts et al 2005, by alanine-scanning mutagenesis. For these studies, TM3 residues were mutated to Ala (or substitution of Ala by Gly) to eliminate the side chain interactions.…”

Section: Resultsmentioning

confidence: 99%

“…The DRY motif and surrounding residues have been known to be essential for receptor signaling in various GPCRs (Gether 2000, Yamano et al 2004, Govaerts et al 2005. Mutations in this motif are known to greatly affect the receptor functions by directly disrupting the G protein coupling and disrupting the ionic lock 0 WT Basal cAMP (% WT) V119A N120A I121A D122A N123A V124A I125A D126A S127A V128A I129A C130A S131A S132A L133A L134A A135G I137A C138A S139A L140A L141A S142A I143A A144G V145A 100 L140D L140E L140F L140G L140I L140K L140M L140N L140P L140Q L140R L140S L140T L140V L140W L140Y WT L140C L140D L140E L140F L140G L140I L140K L140M L140N L140P L140Q L140R L140S L140T L140V L140W formed between TM3 and TM6 (reviewed in Gether (2000)).…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in this motif are known to greatly affect the receptor functions by directly disrupting the G protein coupling and disrupting the ionic lock 0 WT Basal cAMP (% WT) V119A N120A I121A D122A N123A V124A I125A D126A S127A V128A I129A C130A S131A S132A L133A L134A A135G I137A C138A S139A L140A L141A S142A I143A A144G V145A 100 L140D L140E L140F L140G L140I L140K L140M L140N L140P L140Q L140R L140S L140T L140V L140W L140Y WT L140C L140D L140E L140F L140G L140I L140K L140M L140N L140P L140Q L140R L140S L140T L140V L140W formed between TM3 and TM6 (reviewed in Gether (2000)). In the MC4R, mutations of the DRY motif resulted in profoundly impaired ligand binding and receptor signaling properties (Yamano et al 2004). Previous studies mutating D146 into Ala resulted in significantly increased basal cAMP activity.…”

Section: Discussionmentioning

confidence: 99%

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Functions of transmembrane domain 3 of human melanocortin-4 receptor

Mo¹,

Yang²,

Tao³

2012

Journal of Molecular Endocrinology

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The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critical for maintaining energy homeostasis. Transmembrane domain 3 (TM3) of MC4R contains residues that were suggested to be essential in ligand binding and signaling. Several MC4R mutations in TM3 are associated with human obesity. To gain a better understanding of the functions of TM3, we analyzed the functions of 26 residues in TM3 using alanine-scanning mutagenesis. We showed that all mutants had normal cell-surface expression. Four mutants were defective in ligand binding and signaling and six mutants had normal ligand binding but impaired cAMP production. L140A had increased basal cAMP level. To further characterize the function of L140, we generated 17 additional L140 mutants. Fifteen L140 mutants had significantly decreased cell-surface expression, with L140R and L140V expressed normally. Ten L140 mutants had increased basal cAMP activities. Four L140 mutants were defective in ligand-stimulated cAMP generation. Interestingly, with the ERK1/2 pathway, we showed that nine constitutively active mutants had similar levels of basal pERK1/2 as that of WT, and two signaling defective mutants had similar levels of pERK1/2 as that of WT upon agonist stimulation, different from their cAMP signaling properties, suggesting biased signaling in these mutant receptors. In summary, we identified 13 residues in TM3 that were essential for ligand binding and/or signaling. Moreover, L140 was critical for locking MC4R in inactive conformation and several mutants showed biased signaling in cAMP and ERK1/2 signaling pathways.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Functions of transmembrane domain 3 of human melanocortin-4 receptor

Mo¹,

Yang²,

Tao³

2012

Journal of Molecular Endocrinology

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“…With respect to the accuracy of annotation, 3 of the 22 residues classified as important have been experimentally implicated in receptor function: D90 (involved in AGRP inverse agonism) (47) and D146 and R147 located within the DRY motif (50). For 7 others (of the 22), experimental mutagenesis studies report functional effects (N62S, Y157S, R165A/Q/W, A219V, W258A, C271A/Y/R, and P299H; Supplemental Table 1).…”

Section: Discussionmentioning

confidence: 99%

In silico mutagenesis: a case study of the melanocortin 4 receptor

et al. 2009

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The melanocortin 4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) and a key molecule in the regulation of energy homeostasis. At least 159 substitutions in the coding region of human MC4R (hMC4R) have been described experimentally; over 80 of those occur naturally, and many have been implicated in obesity. However, assessment of the presumably functionally essential residues remains incomplete. Here we have performed a complete in silico mutagenesis analysis to assess the functional essentiality of all possible nonnative point mutants in the entire hMC4R protein (332 residues). We applied SNAP, which is a method for quantifying functional consequences of single amino acid (AA) substitutions, to calculate the effects of all possible substitutions at each position in the hMC4R AA sequence. We compiled a mutability score that reflects the degree to which a particular residue is likely to be functionally important. We performed the same experiment for a paralogue human melanocortin receptor (hMC1R) and a mouse orthologue (mMC4R) in order to compare computational evaluations of highly related sequences. Three results are most salient: 1) our predictions largely agree with the available experimental annotations; 2) this analysis identified several AAs that are likely to be functionally critical, but have not yet been studied experimentally; and 3) the differential analysis of the receptors implicates a number of residues as specifically important to MC4Rs vs. other GPCRs, such as hMC1R.—Bromberg, Y., Overton, J., Vaisse, C., Leibel, R. L., Rost, B. In silico mutagenesis: a case study of the melanocortin 4 receptor.

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“…This is an important observation, because it is known that the G protein is coupled to the highly conserved DRY-motif (an ASP-ARG-TYR sequence) located at the end of helix 3. The DRY-motif is believed to play an important role in protein activation (17). Thus, the motions observed with respect to helix 3 provide a strong indication that such motions are involved upon in protein activation.…”

Section: Role Of Water In Mediating the Dynamics Of The Interhelical mentioning

confidence: 99%

Dynamic behavior of fully solvated β2-adrenergic receptor, embedded in the membrane with bound agonist or antagonist

Spijker

Vaidehi

Freddolino

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Recently we predicted the 3D structure of the human ␤2-adrenergic receptor (␤2AR) and of the binding site of several agonists and antagonists to ␤2AR. These predictions (MembStruk and HierDock) included no explicit water and only a few lipid molecules. Here we include explicit H 2O and an infinite lipid bilayer membrane in molecular dynamics (MD) simulations of three systems: apo-␤2AR, epinephrine-bound ␤2AR, and butoxamine-bound ␤2AR (epinephrine is an endogenous agonist, and butoxamine is a ␤2AR selective antagonist). The predicted structures for apo-␤2AR and butoxamine-␤2AR are stable in MD, but in epinephrine-␤2AR, extracellular water trickles into the binding pocket to mediate hydrogen bonding between the catechol of epinephrine and Ser-204 on helix 5. The epinephrine-␤2AR structure shows dynamic flexibility with small, piston-like movements of helices 3 and 6 and transient interhelical hydrogen bonding between Ser-165 on transmembrane 4 and Ser-207 on transmembrane 5. These couplings and motions may play a role in protein activation. The apo-␤2AR shows less dynamic flexibility, whereas the antagonist-␤2AR structure is quite rigid. This MD validation of the structure predictions for G protein-coupled receptors in explicit lipid and water suggests that these methods can be trusted for studying the mechanism of activation and the design of subtype-specific agonists and antagonists.

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The Role of the DRY Motif of Human MC4R for Receptor Activation

Cited by 11 publications

References 15 publications

Functions of transmembrane domain 3 of human melanocortin-4 receptor

Functions of transmembrane domain 3 of human melanocortin-4 receptor

In silico mutagenesis: a case study of the melanocortin 4 receptor

Dynamic behavior of fully solvated β2-adrenergic receptor, embedded in the membrane with bound agonist or antagonist

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