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Distinct Agonist Regulation of Muscarinic Acetylcholine M2-M3 Heteromers and Their Corresponding Homomers.

Aslanoglou D, Alvarez-Curto E, Marsango S, Milligan G - J. Biol. Chem. (2015)

Bottom Line: In this setting occupancy of the receptors with a muscarinic antagonist was without detectable effect on any of the muscarinic oligomers.However, selective agonist occupancy of the M2 receptor resulted in enhanced M2-M2 homomer interactions but decreased M2-M3 heteromer interactions.By contrast, selective activation of the M3 RASSL receptor did not significantly alter either M3-M3 homomer or M2-M3 heteromer interactions.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.

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Concurrent detection of the presence hM2WT homomers and hM2WT-hM3RASSL heteromers and their regulation by carbachol. Cells as in Fig. 9 induced to co-express VSV-SNAP-hM2WT and HA-CLIP-hM3RASSL were incubated with a combination of each of SNAP-Lumi4Tb, CLIP-Red, and SNAP-Green (A, B) and htrFRET signal measured at both 665 nm (SNAP-Lumi4Tb to CLIP-Red (hM2WT-hM3RASSL heteromer)) (A) and 520 nm (SNAP-Lumi4Tb to SNAP-Green (hM2WT homomer)) (B). Treatment with carbachol but not atropine reduced the hM2WT-hM3RASSL heteromer signal (A) and concurrently increased the hM2WT homomer signal (B). Equivalent studies used a combination of CLIP-Lumi4Tb and both SNAP-Green and CLIP-Red (C, D). Treatment with carbachol only reduced the hM2WT-hM3RASSL heteromer (520 nm) signal (C) while neither carbachol, atropine nor CNO had any effect on the hM3RASSL homomer (665 nm) signal (D). Data represent means ± S.E., n =3. Statistical significance as follows: *, p < 0.05, **, p < 0.001, and ***, p < 0.0001 when compared with vehicle.
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Figure 10: Concurrent detection of the presence hM2WT homomers and hM2WT-hM3RASSL heteromers and their regulation by carbachol. Cells as in Fig. 9 induced to co-express VSV-SNAP-hM2WT and HA-CLIP-hM3RASSL were incubated with a combination of each of SNAP-Lumi4Tb, CLIP-Red, and SNAP-Green (A, B) and htrFRET signal measured at both 665 nm (SNAP-Lumi4Tb to CLIP-Red (hM2WT-hM3RASSL heteromer)) (A) and 520 nm (SNAP-Lumi4Tb to SNAP-Green (hM2WT homomer)) (B). Treatment with carbachol but not atropine reduced the hM2WT-hM3RASSL heteromer signal (A) and concurrently increased the hM2WT homomer signal (B). Equivalent studies used a combination of CLIP-Lumi4Tb and both SNAP-Green and CLIP-Red (C, D). Treatment with carbachol only reduced the hM2WT-hM3RASSL heteromer (520 nm) signal (C) while neither carbachol, atropine nor CNO had any effect on the hM3RASSL homomer (665 nm) signal (D). Data represent means ± S.E., n =3. Statistical significance as follows: *, p < 0.05, **, p < 0.001, and ***, p < 0.0001 when compared with vehicle.

Mentions: As an extension to these studies we attempted to identify concurrently in the same cells both homo- and hetero-interactions involving VSV-SNAP-hM2WT. SNAP- and CLIP-Lumi4Tb have broad emission spectra. As such, upon excitation at 337 nm they can potentially transfer energy to both Green (with htrFRET output at 520 nm) and Red (with htrFRET output at 665 nm) energy acceptors. This potentially allows concurrent dual color detection of multiple interactions of the energy donor-tagged receptor. We, therefore, initially added a mixture of SNAP-Lumi4Tb and both CLIP-Red and SNAP-Green to cells induced to co-express VSV-SNAP-hM2WT and HA-CLIP-hM3RASSL. Such studies were indeed able to identify interactions of the energy donor-labeled hM2WT with both energy acceptor labeled hM2WT and hM3RASSL receptors concurrently (Fig. 10, A and B). Moreover, as in the individual htrFRET experiments reported above, concurrent analysis of the two distinct interactions of the energy donor-labeled hM2WT receptor showed an equivalent carbachol-mediated decrease in hM2WT-hM3RASSL heteromeric interactions (Fig. 10A) and increase in hM2WT-hM2WT homomeric interactions (Fig. 10B). Once again, the muscarinic antagonist atropine was without effect (Fig. 10, A and B). Finally, in cells induced to co-express the hM2WT and hM3RASSL receptors, labeling of hM3RASSL with the energy donor CLIP-Lumi4Tb and proportions of both hM2WT and hM3RASSL respectively with SNAP-Green and CLIP-Red, the hM2WT-hM3RASSL heteromeric interactions were again specifically decreased by treatment with carbachol (Fig. 10C). By contrast hM3RASSL homo-interactions were once more unperturbed by addition of any of CNO, carbachol, or atropine (Fig. 10D).


Distinct Agonist Regulation of Muscarinic Acetylcholine M2-M3 Heteromers and Their Corresponding Homomers.

Aslanoglou D, Alvarez-Curto E, Marsango S, Milligan G - J. Biol. Chem. (2015)

Concurrent detection of the presence hM2WT homomers and hM2WT-hM3RASSL heteromers and their regulation by carbachol. Cells as in Fig. 9 induced to co-express VSV-SNAP-hM2WT and HA-CLIP-hM3RASSL were incubated with a combination of each of SNAP-Lumi4Tb, CLIP-Red, and SNAP-Green (A, B) and htrFRET signal measured at both 665 nm (SNAP-Lumi4Tb to CLIP-Red (hM2WT-hM3RASSL heteromer)) (A) and 520 nm (SNAP-Lumi4Tb to SNAP-Green (hM2WT homomer)) (B). Treatment with carbachol but not atropine reduced the hM2WT-hM3RASSL heteromer signal (A) and concurrently increased the hM2WT homomer signal (B). Equivalent studies used a combination of CLIP-Lumi4Tb and both SNAP-Green and CLIP-Red (C, D). Treatment with carbachol only reduced the hM2WT-hM3RASSL heteromer (520 nm) signal (C) while neither carbachol, atropine nor CNO had any effect on the hM3RASSL homomer (665 nm) signal (D). Data represent means ± S.E., n =3. Statistical significance as follows: *, p < 0.05, **, p < 0.001, and ***, p < 0.0001 when compared with vehicle.
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Figure 10: Concurrent detection of the presence hM2WT homomers and hM2WT-hM3RASSL heteromers and their regulation by carbachol. Cells as in Fig. 9 induced to co-express VSV-SNAP-hM2WT and HA-CLIP-hM3RASSL were incubated with a combination of each of SNAP-Lumi4Tb, CLIP-Red, and SNAP-Green (A, B) and htrFRET signal measured at both 665 nm (SNAP-Lumi4Tb to CLIP-Red (hM2WT-hM3RASSL heteromer)) (A) and 520 nm (SNAP-Lumi4Tb to SNAP-Green (hM2WT homomer)) (B). Treatment with carbachol but not atropine reduced the hM2WT-hM3RASSL heteromer signal (A) and concurrently increased the hM2WT homomer signal (B). Equivalent studies used a combination of CLIP-Lumi4Tb and both SNAP-Green and CLIP-Red (C, D). Treatment with carbachol only reduced the hM2WT-hM3RASSL heteromer (520 nm) signal (C) while neither carbachol, atropine nor CNO had any effect on the hM3RASSL homomer (665 nm) signal (D). Data represent means ± S.E., n =3. Statistical significance as follows: *, p < 0.05, **, p < 0.001, and ***, p < 0.0001 when compared with vehicle.
Mentions: As an extension to these studies we attempted to identify concurrently in the same cells both homo- and hetero-interactions involving VSV-SNAP-hM2WT. SNAP- and CLIP-Lumi4Tb have broad emission spectra. As such, upon excitation at 337 nm they can potentially transfer energy to both Green (with htrFRET output at 520 nm) and Red (with htrFRET output at 665 nm) energy acceptors. This potentially allows concurrent dual color detection of multiple interactions of the energy donor-tagged receptor. We, therefore, initially added a mixture of SNAP-Lumi4Tb and both CLIP-Red and SNAP-Green to cells induced to co-express VSV-SNAP-hM2WT and HA-CLIP-hM3RASSL. Such studies were indeed able to identify interactions of the energy donor-labeled hM2WT with both energy acceptor labeled hM2WT and hM3RASSL receptors concurrently (Fig. 10, A and B). Moreover, as in the individual htrFRET experiments reported above, concurrent analysis of the two distinct interactions of the energy donor-labeled hM2WT receptor showed an equivalent carbachol-mediated decrease in hM2WT-hM3RASSL heteromeric interactions (Fig. 10A) and increase in hM2WT-hM2WT homomeric interactions (Fig. 10B). Once again, the muscarinic antagonist atropine was without effect (Fig. 10, A and B). Finally, in cells induced to co-express the hM2WT and hM3RASSL receptors, labeling of hM3RASSL with the energy donor CLIP-Lumi4Tb and proportions of both hM2WT and hM3RASSL respectively with SNAP-Green and CLIP-Red, the hM2WT-hM3RASSL heteromeric interactions were again specifically decreased by treatment with carbachol (Fig. 10C). By contrast hM3RASSL homo-interactions were once more unperturbed by addition of any of CNO, carbachol, or atropine (Fig. 10D).

Bottom Line: In this setting occupancy of the receptors with a muscarinic antagonist was without detectable effect on any of the muscarinic oligomers.However, selective agonist occupancy of the M2 receptor resulted in enhanced M2-M2 homomer interactions but decreased M2-M3 heteromer interactions.By contrast, selective activation of the M3 RASSL receptor did not significantly alter either M3-M3 homomer or M2-M3 heteromer interactions.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.

Show MeSH