A Novel Interaction between Adrenergic Receptors and the α-Subunit of Eukaryotic Initiation Factor 2B

Klein, Uwe; Ramirez, M T; Kobilka, Brian K.; Zastrow, Mark von

doi:10.1074/jbc.272.31.19099

Cited by 72 publications

(46 citation statements)

References 32 publications

(24 reference statements)

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“…The colocalization of M 4 and eEF1A2 in the neuropil supports the hypothesis that the interaction between M 4 and eEF1A2 may be a mechanism for direct muscarinic modulation of dendritic translation. Furthermore, adrenergic receptors have been demonstrated to interact with the ␣ subunit of eIF2B, suggesting that other GPCRs may directly regulate translation (40). Because agonist stimulation had no effect on the physical association between eEF1A2 and M 4 in our co-immunoprecipitation assay, it is still unclear what activates the eEF1A2 GEF activity of M 4 .…”

Section: Discussionmentioning

confidence: 67%

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

McClatchy¹,

Knudsen²,

Clark³

et al. 2002

Journal of Biological Chemistry

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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 ...

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Section: Discussionmentioning

confidence: 67%

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

McClatchy¹,

Knudsen²,

Clark³

et al. 2002

Journal of Biological Chemistry

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“…For the in vivo interaction studies, specific, high a m t y antibodies suitable for imrnunoprecipitation and directed against the dopamine D4 receptor have been developed (HofianLaRoche and Allelix/Hoehst). These antibodies in combination with the adenoviral expression techniques developed in our lab, to express high levels of the dopamine D4 receptor, in addition to recently developed imniunoprecipitation protocols that have been successfully used to dernonstrate GPCR interaction with intracellukir proteins (Kinoor et al, 1998;Klein et al, 1997) In trying to demonstrate the D4/SH3 interaction in vitro, we tried to express a GST fusion protein of the full length D4 receptor and a fragment encoding the third intracellular loop. The D4 receptor and the third intracellular loop fragment were resistant to expression in the GST-fusion protein system when expressed in bacteriai cells.…”

Section: Future Researchmentioning

confidence: 99%

SH3 Binding Domains in the Dopamine D4 Receptor

Oldenhof

Vickery²,

Anafi³

et al. 1998

Biochemistry

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The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2−SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor−G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

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“…One domain that is conserved in both ␣ 2 -and ␤ 2 -adrenergic receptors, the carboxyl-terminal DFRXXFXXXL motif, interacts with the ␣-subunit of the eukaryotic initiation factor 2B (9). This protein interaction enhances the ␤ 2 -adrenergic receptor-mediated activation of adenylyl cyclase (9). The glutamate/dileucine sequence motif conserved in many G protein-coupled receptors is involved in the cell surface transport of receptors (21).…”

Section: Resultsmentioning

confidence: 99%

“…These interactions were first inferred from the functional effects of cytoplasmic proteins on receptor signaling and desensitization and were later confirmed by biochemical observation of the binding of the protein with receptor (5)(6)(7)(8). Very recently, however, several unexpected interactions between cytoplasmic proteins and receptors have been observed; for instance, the adrenergic receptor interacts with the ␣-subunit of the eukaryotic initiation factor 2B (9) and with the Na ϩ /H ϩ -exchange regulatory factor (10). These raise the possibility that receptors may interact with other types of cellular proteins that could play unanticipated roles in regulating the function of the receptor.…”

mentioning

confidence: 96%

Interaction of the α1B-Adrenergic Receptor with gC1q-R, a Multifunctional Protein

Xu¹,

Hirasawa

Shinoura

et al. 1999

Journal of Biological Chemistry

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gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the ␣ 1B -adrenergic receptor (173 amino acids, amino acids 344 -516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369 -378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with fulllength ␣ 1B -adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the ␣ 1B -adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the ␣ 1B -adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that coexpressed ␣ 1B -adrenergic receptors and gC1q-R, most of the ␣ 1B -adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the ␣ 1B -adrenergic receptors.G protein-coupled receptors interact with several classes of cytoplasmic proteins including heterotrimeric G proteins, kinases, phosphatases, and arrestins, and the binding of cytoplasmic protein with the receptor regulates receptor signaling (1-4). These interactions were first inferred from the functional effects of cytoplasmic proteins on receptor signaling and desensitization and were later confirmed by biochemical observation of the binding of the protein with receptor (5-8). Very recently, however, several unexpected interactions between cytoplasmic proteins and receptors have been observed; for instance, the adrenergic receptor interacts with the ␣-subunit of the eukaryotic initiation factor 2B (9) and with the Na ϩ /H ϩ -exchange regulatory factor (10). These raise the possibility that receptors may interact with other types of cellular proteins that could play unanticipated roles in regulating the function of the receptor.We conducted a search for novel proteins that interact with the ␣ 1B -adrenergic receptor, specifically focusing on the carboxyl-terminal cytoplasmic domain, because mutations within this domain have pleiotropic effects on receptor physiology (11)(12)(13)(14). Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R 1 interacts with ␣ 1B -adrenergic receptors in vitro and in vivo through the specific site and that in cells that co-express ␣ 1B -adrenergic receptors and gC1q-R, the subcellular localization of ␣ 1B -adrenergic receptors is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as ...

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A Novel Interaction between Adrenergic Receptors and the α-Subunit of Eukaryotic Initiation Factor 2B

Cited by 72 publications

References 32 publications

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

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

SH3 Binding Domains in the Dopamine D4 Receptor

Interaction of the α1B-Adrenergic Receptor with gC1q-R, a Multifunctional Protein

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