Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans.
Nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory neurotransmission in neurons and muscles. To identify nAChR accessory proteins, which may regulate their expression or function, we performed tandem affinity purification of the levamisole-sensitive nAChR from Caenorhabditis elegans, mass spectrometry of associated components, and RNAi-based screening for effects on in vivo nicotine sensitivity. Among the proteins identified was the calcineurin A subunit TAX-6, which appeared to function as a negative regulator of nAChR activity. We also identified five proteins not previously linked to nAChR function, whose inactivation conferred nicotine resistance, implicating them as positive regulators of nAChR activity. Of these, the copine NRA-1 colocalized with the levamisole receptor at neuronal and muscle plasma membranes, and, when mutated, caused reduced synaptic nAChR expression. Loss of SOC-1, which acts in receptor tyrosine kinase (RTK) signaling, also reduced synaptic levamisole receptor levels, as did mutations in the fibroblast growth factor receptor EGL-15, and another RTK, CAM-1. Thus, tandem affinity purification is a viable approach to identify novel proteins regulating neurotransmitter receptor activity or expression in model systems like C. elegans.
Gottschalk
Alexander
A
Institute for Biochemistry, Goethe-University, Frankfurt, Germany. a.gottschalk@em.uni-frankfurt.de
Almedom
Ruta B
RB
Schedletzky
Thorsten
T
Anderson
Scott D
SD
Yates
John R
JR
Schafer
William R
WR
D000345:Affinity Labels; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D017930:Genes, Reporter; D007978:Levamisole / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D034622:RNA Interference; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism; D013997:Time Factors
D000345:Affinity Labels; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D018722:Nicotinic Agonists; D011950:Receptors, Cholinergic; D017468:Receptors, Fibroblast Growth Factor; D011978:Receptors, Nicotinic; C110203:copine; D007978:Levamisole; D009538:Nicotine