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Muscarinic M4 receptor recycling requires a motif in the third intracellular loop

Yuichi Hashimoto1, Kanoko Morisawa, Hiroyuki Saito

  • 1Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan. yuichi.hashimoto@gakushuin.ac.jp

The Journal of Pharmacology and Experimental Therapeutics
|March 14, 2008
PubMed

Insights

Specific regions within the M4 muscarinic acetylcholine receptor's third intracellular loop are crucial for its internalization and recycling. The Val(373)-Ala(393) sequence is essential for recycling, while Leu(272)-Arg(338) also plays a role in internalization.

Area of Science:

  • Pharmacology
  • Molecular Biology
  • Cell Biology

Background:

  • Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors involved in various physiological processes.
  • Receptor trafficking, including internalization and recycling, is critical for regulating cellular responses to agonists.
  • The M4 subtype (M4 receptor) plays a role in central nervous system functions, and its trafficking is not fully understood.

Purpose of the Study:

  • To identify specific amino acid sequences within the third intracellular loop (i3) of the M4 receptor that mediate its agonist-dependent internalization and subsequent recycling.
  • To elucidate the functional roles of different M4 receptor i3 subregions in receptor trafficking dynamics.

Main Methods:

  • Site-directed mutagenesis was used to create M4 receptor mutants with partial deletions in the i3 region.
  • Internalization and recycling assays were performed using transiently transfected human embryonic kidney 293-tsA201 cells.
  • Functional characterization of chimeric receptors involving M4 and M2 receptor sequences was conducted.

Main Results:

  • Mutants M4del-K(235)-K(240), M4del-T(241)-K(271), and M4del-W(339)-N(372) exhibited similar internalization and recycling patterns to the wild-type M4 receptor.
  • The M4del-L(272)-R(338) mutant showed reduced internalization (50%) but retained recycling capacity.
  • The M4del-V(373)-A(393) mutant also displayed reduced internalization (50%) and was deficient in recycling.
  • Grafting the Val(373)-Ala(393) sequence onto a recycling-deficient M2 receptor mutant restored significant internalization and recycling.

Conclusions:

  • The Leu(272)-Arg(338) and Val(373)-Ala(393) regions within the M4 receptor i3 are involved in mediating receptor internalization.
  • The Val(373)-Ala(393) sequence is indispensable for the recycling of internalized M4 receptors back to the cell surface.
  • Other regions of the i3 loop are dispensable for M4 receptor internalization and recycling.

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