Comparison of the pharmacological antagonism of M2 and M3 muscarinic receptors expressed in isolation and in combination

Griffin, Michael T.; Hsu, Jake Ching-Hsuan; Shehnaz, Darakhshanda; Ehlert, Frederick J.

doi:10.1016/s0006-2952(03)00068-6

Cited by 20 publications

(24 citation statements)

References 16 publications

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“…(b) Some studies have argued against the existence of mAChR heterodimers. In particular, the presence of M 2 /M 3 constitutive heterodimers has been challenged by recent reports and is still a matter of controversy (9,(35)(36)(37). (c) M 2 and M 3 mAChR regulation has typically been examined using cell systems expressing a single mAChR subtype.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

Goin

Nathanson

2006

Journal of Biological Chemistry

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Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo-and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M 1 , M 2 , and M 3 mAChR can form constitutive homo-and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M 1 , M 2 , or M 3 mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M 1 /M 2 , M 2 /M 3 , and M 1 /M 3 mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo-and heterodimers. When expressed in JEG-3 cells, the M 2 receptor exhibits much higher susceptibility than the M 3 receptor to agonistinduced down-regulation. Coexpression of M 3 mAChR with increasing amounts of the M 2 subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M 3 , suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.Over the last decade, many studies have shown broad evidence that G protein-coupled receptors (GPCR) 3 interact with one another to form constitutive homo-or hetero-oligomers. GPCR dimerization has been implicated in many aspects of receptor pharmacology, such as maturation, ligand binding, activation, signaling, desensitization, endocytosis, and trafficking (1-3). In the early 1990s, several studies showed that muscarinic acetylcholine receptors (mAChR), like other GPCR, might be arranged in dimeric or oligomeric complexes. Complex binding curves for both muscarinic agonists and antagonists were interpreted as evidence for the existence of multiple states of affinity, which is consistent with the notion of binding sites located on dimeric receptor molecules (4, 5). More recent studies on mAChR have shown that such heterogeneity of affinity should rather be attributed to nucleotide-sensitive cooperative effects between interacting sites (6, 7). Because there seems to be one binding site per receptor molecule, cooperativity suggests that mAChR are actually oligomers formed by two or more interacting sites (6).Additional pharmacological evidence that mAChR can form dimers was provided by Maggio et al. (8), who used chimeric ␣ 2 -adrenergic/M 3 muscarinic receptors composed of the first five transmembrane domains (TMD) of one receptor and the last two TMD of the other. No binding or signaling was detected when either fusion protein was expressed alone, but coexpression of both chimeras rescued binding activity and signaling properties of both adrenergic and muscarinic ligands. These results were interpreted as intermolecular interactions between two inactive receptors now eng...

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Section: Discussionmentioning

confidence: 99%

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

Goin

Nathanson

2006

Journal of Biological Chemistry

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“…Atropine-treated cells were washed three times with F-12K (500 l), with a 20-min incubation between each wash in a humidified incubator. After the third and final wash, cells were incubated for various periods of time for up to 8 h in a humidified incubator set at 37°C in an atmosphere of 5% CO 2 /95% air, then used in intact, whole- (Griffin et al, 2003), and radioactivity was counted by use of a Beckman LS 6500 scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

“…To (Griffin et al, 2003), and radioactivity was counted by use of a Beckman LS6500 scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M₁Acetylcholine Receptors Affects Plasma Membrane Expression

Sawyer¹,

Ehlert²,

Shults³

2009

J Pharmacol Exp Ther

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We investigated the functional role of a conserved motif, F(x) 6 LL, in the membrane proximal C-tail of the human muscarinic M 1 (hM 1 ) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence 423 FRDTFRLLL 431 . Wild-type and mutant hM 1 receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [ play a role in folding hM 1 receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V 46 and L 47 , that, when mutated, reduce the plasma membrane expression of hM 1 receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM 1 receptors to achieve a transport-competent state.

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“…Cells were washed extensively on ice with ice-cold PBS (three 500-l washes) to remove carbachol. Intact cell binding assays were then performed using either a single concentration 3 H]QNB was recovered as described previously (Griffin et al, 2003), and radioactivity was counted using a Beckman LS 6500 scintillation counter. Saturation binding assays were also performed on washed, intact CHO cells transiently expressing either wild-type or mutant hM 1 receptors.…”

Section: Methodsmentioning

confidence: 99%

Cysteine Pairs in the Third Intracellular Loop of the Muscarinic M₁Acetylcholine Receptor Play a Role in Agonist-Induced Internalization

Sawyer

Ehlert²,

Shults³

2007

J Pharmacol Exp Ther

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We determined the functional role of a small domain in the third intracellular loop of the human muscarinic M 1 (hM 1 ) receptor. Using site-directed mutagenesis, several mutant hM 1 receptors were made possessing either a deletion or point mutations within the third intracellular loop domain 252 PETPPGRCCRCC 263 . Wild-type and mutant hM 1 receptors were transiently expressed in Chinese hamster ovary cells, and the effects of each mutation on radioligand binding, agonist-mediated phosphoinositide hydrolysis, and agonist-induced internalization were determined. The mutant receptors exhibited a modest reduction in affinity for [ 3 H]N-methylscopolamine (pK D ϭ ϳ9.0) and a moderately increased binding capacity relative to the wild-type receptor. This moderate increase in binding capacity was associated with small increases in the maximal response and potency of carbachol for eliciting phosphoinositide hydrolysis through the mutant receptors (pEC 50 ϭ ϳ5.5) relative to wild-type (pEC 50 ϭ 5.35 Ϯ 0.05). In contrast, carbachol-induced internalization of mutant hM 1 receptors possessing either C259A/C260A or C262A/C263A or both double point mutations was significantly reduced compared to the wild-type hM 1 receptor. Of the hM 1 receptor mutants tested, those possessing a C262D/C263D double point mutation had the least carbachol-induced internalization. The desensitization and down-regulation of receptors possessing either Cys/Ala or Cys/Asp double point mutations were similar to those observed for the wild-type hM 1 receptor. Collectively, these observations suggest that Cys pairs Cys259/Cys260 and Cys262/Cys263 play an important role in the agonist-induced internalization of hM 1 receptors.

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Comparison of the pharmacological antagonism of M2 and M3 muscarinic receptors expressed in isolation and in combination

Cited by 20 publications

References 16 publications

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M₁Acetylcholine Receptors Affects Plasma Membrane Expression

Cysteine Pairs in the Third Intracellular Loop of the Muscarinic M₁Acetylcholine Receptor Play a Role in Agonist-Induced Internalization

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Comparison of the pharmacological antagonism of M2 and M3 muscarinic receptors expressed in isolation and in combination

Cited by 20 publications

References 16 publications

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1Acetylcholine Receptors Affects Plasma Membrane Expression

Cysteine Pairs in the Third Intracellular Loop of the Muscarinic M1Acetylcholine Receptor Play a Role in Agonist-Induced Internalization

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A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M₁Acetylcholine Receptors Affects Plasma Membrane Expression

Cysteine Pairs in the Third Intracellular Loop of the Muscarinic M₁Acetylcholine Receptor Play a Role in Agonist-Induced Internalization