Deletion analysis of the m4 muscarinic acetylcholine receptor

Abstract

In order to investigate whether coupling to and/or activation of guanine-nucleotide-binding proteins (G proteins) is involved in agonist-induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des-(264-394)mAChRl was constructed that lacks a substantial portion of the putative third intracellular loop. The wild-type receptor and des-(264 -394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist-binding affinities. Consistent with the reported importance of the third cytoplasmic loop for G, protein activation, the des-(264-394)mAChR showed a drastically reduced potential to mediate agonist-induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to GI proteins was not impaired, as demonstrated by a similar guaninenucleotide-sensitive and pertussis-toxin-sensitive high-affinity agonist-receptor binding for both mAChRs. In contrast, des-(264-394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of G, proteins rather than ineffective coupling to GI proteins. Internalization of wild-type receptor and des-(264 -394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des-(264-394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild-type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in G,-protein coupling and activation are not identical and that activation of, but not coupling to, G, proteins regulates m4 mAChR internalization.Muscarinic acetylcholine receptors (mAChRs) belong to the superfamily of seven transmembrane receptors which activate signal-transduction pathways through their interaction with guanine-nucleotide-binding regulatory proteins (G proteins). The mAChR family consists of five receptors of which the ml, m3 and m5 receptor subtypes are efficiently coupled to stimulation of phosphoinositide-hydrolyzing phospholipase C via pertussis toxin (PTx)-insensitive G, proteins, and m2 and m4 receptors which are preferentially coupled to the inhibition of adenylyl cyclase and to other effectors via PTxsensitive Gi and Go proteins [l]. Studies involving mutations of the ml, m2 and m3 mAChRs have led to the conlusion that third cytoplasmic loop regions are involved in G proteinspecificity. Exchanging third cytoplasmic loops among m l and m2 mAChRs [2] and m2 and m3 mAChRs [3, 41 altered the preferred second messenger pathways between the control of phospholipase C and adenylyl cyclase activities. In Correspondence to C . J. van Koppen,

Desensitization and Internalization of the m2 Muscarinic Acetylcholine Receptor Are Directed by Independent Mechanisms

“…Deletions of the C terminus of the m1 mAChR had no effect on receptor internalization, while mutations in the i3 loop reduced internalization (63). In addition, deletion of the i3 loop of the m4 mAChR slowed the rate of internalization initially, but by 1 h internalization was equivalent to that seen in wild-type receptor (64). A slight reduction in m2 mAChR internalization was observed when several serine/threonine residues within the region deleted in our studies were mutated to alanine (47).…”

“…Studies of the angiotensin II receptor (59), the ␤ 2 -AR (60), the parathyroid hormone receptor (61), and the ␣ 1B -adrenergic receptor (62) indicate that the C termini of these proteins are involved in internalization while the i3 loop of several subtypes of mAChRs are the likely regions for internalization control of these receptors (47,63,64). Deletions of the C terminus of the m1 mAChR had no effect on receptor internalization, while mutations in the i3 loop reduced internalization (63).…”

Novel Interaction between the M4 Muscarinic Acetylcholine Receptor and Elongation Factor 1A2

“…Specifically, the portion of the M 4 i3 loop used in our initial brain pull-down assays has been reported to be involved in the GEF activity of G i/o (31,32). To test our hypothesis, we performed a nucleotide exchange assay using the fluorescent GDP analog, mantGDP, and recombinant eEF1A2.…”

“…Upon activation, the mAChR i3 loop activates heterotrimeric G-proteins by acting as a guanine exchange factor (GEF), which stimulates the release of GDP allowing GTP to bind (31,32). Hence, we hypothesized that M 4 may activate eEF1A2 similarly by regulating the nucleotide exchange rate of eEF1A2.…”

Role of the third intracellular loop in the subtype-specific internalization and recycling of muscarinic M2 and M4 receptors

“…Extensive studies on internalization of GPCRs, particularly on β 2 -adrenergic receptors, have indicated that the internalization involves the following processes: (A) agonist-dependent phosphorylation of receptors by G proteincoupled receptor kinases (GRKs), (B) binding of cytosolic proteins termed as arrestin to phosphorylated receptors, (C) binding of clathrin and other proteins to arrestin-bound receptors, and (D) pinching off of clathrin-coated vesicles including arrestinbound receptors from plasma membranes by action of a protein termed as dynamin with GTPase activity (see a review, 2). By using mutants with a deletion in the i3 loop, several reports have indicated that the i3 loops are involved in internalization of M1 (14,16), M2 (17,26), M3 (17), and M4 (7,30) receptors. Agonist-nalization.…”