Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors

Justin R King,Jacob C Nordman,Samuel P Bridges,Ming-Kuan Lin,Nadine Kabbani

doi:10.1074/jbc.m115.647040

Justin R King, Jacob C Nordman + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m115.647040

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Abstract

α7 nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic transmission and inflammation. In response to ligands, this receptor channel opens to conduct cations into the cell but desensitizes rapidly. In recent studies we show that α7 nAChRs bind signaling proteins such as heterotrimeric GTP-binding proteins (G proteins). Here, we demonstrate that direct coupling of α7 nAChRs to G proteins enables a downstream calcium signaling response that can persist beyond the expected time course of channel activation. This process depends on a G protein-binding cluster (GPBC) in the M3-M4 loop of the receptor. A mutation of the GPBC in the α7 nAChR (α7345-348A) abolishes interaction with Gαq as well as Gβγ while having no effect on receptor synthesis, cell-surface trafficking, or α-bungarotoxin binding. Expression of α7345-348A, however, did significantly attenuate the α7 nAChR-induced Gαq calcium signaling response as evidenced by a decrease in PLC-β activation and IP3R-mediated calcium store release in the presence of the α7 selective agonist choline. Taken together, the data provides new evidence for the existence of a GPBC in nAChRs serving to promote intracellular signaling.

Highlights

␣7 nicotinic acetylcholine receptors play an important role in synaptic transmission and inflammation
An association between ␣7 nicotinic acetylcholine receptors (nAChRs) and G␤ was observed in the prefrontal cortex and hippocampus, whereas little to no detection of G␣o was seen in the adult brain (Fig. 1A)
We show a similar G protein-binding cluster (GPBC) in ␣7 nAChRs and propose the existence of a GPBC in other

Summary

Experimental Procedures

Animals—Male and female C57BL/6 adult mice (Jackson) were housed on a 12-h light/dark cycle and fed ad libitum. G Protein Binding in nAChRs plates (Life Technologies) coated with either collagen (50 ␮g/ml) or poly-D-lysine (100 ␮g/ml) as described [4, 15]. Plasmids used were: GCaMP5G (Addgene, Cambridge, MA); PH-mCherry (Addgene); human ␣7 in pcDNA3.1 [14], Ric-3 Tetracycline (Abgent, San Diego, CA) at a 1:3 ratio of Ric-3 to ␣7 nAChR cDNA and induced after exposure to 2 ␮g/ml of tetracycline in cell media; the human ␣7 nAChR in pEYFP-C1 (Addgene), which is tagged at the amino terminus of the receptor protein. Proteins were eluted in LDS buffer (Life Technologies), separated onto BisTris gradient gels, and transferred onto nitrocellulose membranes (Invitrogen). Eight cells were imaged per experimental condition and all experiments were performed in triplicate to obtain group averages (n ϭ 8). Data are presented as mean Ϯ S.E. of at least three independent experiments

Results

Discussion

G Protein Coupling Activates a Local Rise in Intracellular