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

Abstract

The activation of the muscarinic acetylcholine receptor (mAChR) family, consisting of five subtypes (M 1 -M 5 ), produces a variety of physiological effects throughout the central nervous system. However, the role of each individual subtype remains poorly understood. To further elucidate signal transduction pathways for specific subtypes, we used the most divergent portion of the subtypes, the intracellular third (i3) loop, as bait to identify interacting proteins. Using a brain pull-down assay, we identify elongation factor 1A2 (eEF1A2) as a specific binding partner to the i3 loop of M 4 , and not to M 1 or M 2 .In addition, we demonstrate a direct interaction between these proteins. In the rat striatum, the M 4 mAChR colocalizes with eEF1A2 in the soma and neuropil. In PC12 cells, endogenous eEF1A2 co-immunoprecipitates with the endogenous M 4 mAChR, but not with the endogenous M 1 mAChR. In our in vitro model, M 4 dramatically accelerates nucleotide exchange of eEF1A2, a GTP-binding protein. This indicates the M 4 mAChR is a guanine exchange factor for eEF1A2. eEF1A2 is an essential GTP-binding protein for protein synthesis. Thus, our data suggest a novel role for M 4 in the regulation of protein synthesis through its interaction with eEF1A2.In the central nervous system, the muscarinic acetylcholine receptors (mAChR) 1 play crucial roles in learning, memory, movement, analgesia, and sleep (1-3). Dysfunction in mAChR signaling has been implicated in brain disorders, including Alzheimer's disease, Parkinson's disease, and schizophrenia (4 -6). The mAChRs belong to the G-protein-coupled receptor (GPCR) superfamily. Upon agonist binding, GPCRs bind and activate heterotrimeric G-proteins, which in turn activate various downstream targets. There are two distinct, well characterized G-protein signaling pathways activated by the five mAChR subtypes. M 1 , M 3 , and M 5 couple to G q , which stimulates phospholipase C-␤, and thus releases calcium from intracellular stores and activates protein kinase C (7, 8). M 2 and M 4 are coupled to G i/o , which regulates adenylate cyclase (9). These subtypes are expressed throughout the brain, and, in some brain regions, multiple subtypes are expressed in individual neurons (10). The diversity of physiological effects and the overlapping expression pattern of the muscarinic receptor subtypes suggest that there are signaling pathways initiated by the mAChRs independent of these two heterotrimeric G-protein pathways.Signaling pathways independent of heterotrimeric G-proteins have been reported throughout the GPCR superfamily (11-14). Evidence supporting this hypothesis stems from the identification of novel binding partners of GPCRs other than the traditional heterotrimeric G-proteins. Within the muscarinic family in particular, there is an especially large body of evidence to suggest the existence of nontraditional signaling pathways. There are many reports of G-protein-independent regulation of ion channels by mAChRs (15-18). Moreover, the M 3 mAChR has been found to associate ...

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

“…Studies indicate that eEF family proteins also bind to neurotransmitter receptors, such as the D3 dopamine and M4 muscarinic receptors, and mediate their trafficking to the plasma membrane (45,46). We have uncovered an association between the ␤2 subunit and eEF2.…”

Muscarinic M4 Receptor Recycling Requires a Motif in the Third Intracellular Loop

“…Dominantnegative (V54D) ␤-arrestin 2 (Ferguson et al, 1996) also did not affect internalization of wild-type M4 receptor. Another candidate for interaction with the sequence might be elongation factor 1A, which was reported to interact with i3 of M4 receptors (McClatchy et al, 2002) and inhibit recycling of M4 receptors (McClatchy et al, 2006). It remains to be elucidated whether elongation factor 1A may interact with the region, Val 373 -Ala 393 , and compete with the putative protein(s) that interact with the region and facilitate the recycling.…”

“…On the other hand, the jpet.aspetjournals.org central part of i3 appears to be responsible for regulation of receptors. The i3 of M2 has been shown to be the site for phosphorylation (Nakata et al, 1994) and for interaction with G␤␥ (Wu et al, 1998); i3 of M3 has been shown to be the site for interaction with proteins, such as ␤-arrestin, G␤␥, casein kinase 1␣, and SET (Wu et al, 1997(Wu et al, , 2000Budd et al, 2000;Simon et al, 2006); i3 of M1 has been shown to be the site for interaction with regulators of G protein signaling (Bernstein et al, 2004); and i3 of M4 has been shown to be the site for interaction with elongation factor (McClatchy et al, 2002).…”

Proteomics Analysis of Rat Brain Postsynaptic Density

“…Indeed, EF-1␣ has been shown to bind and modulate activities of diverse signaling proteins such as calmodulin (77), PLC-␥1 (78) and was shown to interact with muscarinic acetylcholine receptors (79). The role of EF-1␣ in the PSD has not been examined.…”