Localization of the Calcitonin Gene-related Peptide Receptor Complex at the Vertebrate Neuromuscular Junction and Its Role in Regulating Acetylcholinesterase Expression

Abstract

The calcitonin gene-related peptide (CGRP) is released by motor neurons where it exerts both short and long term effects on skeletal muscle fibers. In addition, sensory neurons release CGRP on the surrounding vasculature where it is in part responsible for local vasodilation following muscle contraction. Although CGRPbinding sites have been demonstrated in whole muscle tissue, the type of CGRP receptor and its associated proteins or its cellular localization within the tissue have not been described. Here we show that the CGRPbinding protein referred to as the calcitonin receptorlike receptor is highly concentrated at the avian neuromuscular junction together with its two accessory proteins, receptor activity modifying protein 1 and CGRP-receptor component protein, required for ligand specificity and signal transduction. Using tissue-cultured skeletal muscle we show that CGRP stimulates an increase in intracellular cAMP that in turn initiates down-regulation of acetylcholinesterase expression at the transcriptional level, and, more specifically, inhibits expression of the synaptically localized collagen-tailed form of the enzyme. Together, these studies suggest a specific role for CGRP released by spinal cord motoneurons in modulating synaptic transmission at the neuromuscular junction by locally inhibiting the expression of acetylcholinesterase, the enzyme responsible for terminating acetylcholine neurotransmission.The calcitonin gene-related peptide (CGRP) 1 is a 37-amino acid neuropeptide expressed in a wide variety of neurons in the central and peripheral nervous systems, including motoneurons (reviewed in Refs. 1-4), where it is released upon stimulation (5). In skeletal muscle, CGRP has been shown to potentiate muscle contraction (6), increase the rate of acetylcholine receptor (AChR) desensitization (7-10), increase the numbers of AChR (11), decrease the levels of acetylcholinesterase (AChE) (12)(13)(14), and locally increase the rate of blood flow following muscle contraction (15)(16)(17). Although the presence of CGRP in nerve terminals at the neuromuscular junction has been well documented (18 -21), the localization of CGRP receptors at the synapse has been more difficult to demonstrate because the only available probe was the receptor ligand itself, CGRP. Using 125 I-CGRP as a probe, high affinity CGRP receptors were shown to be expressed on the surfaces of tissuecultured myotubes (22) and present on whole muscle membranes isolated from embryonic or newly hatched chicks (23); however, the latter studies could not differentiate between CGRP receptors on skeletal muscle versus those on the surrounding vasculature. In one autoradiographic study (24), a correlation between 125 I-CGRP and AChE histochemical staining was observed in low power photomicrographs of rat bulbocavernosus muscle, suggesting an accumulation of CGRP receptors at sites of nerve-muscle contact. However, in these studies it was not possible to differentiate between CGRP receptors located in blood vessels or Schwann cells and those on m...

Regulation of a Transcript Encoding the Proline-rich Membrane Anchor of Globular Muscle Acetylcholinesterase

“…In contrast with the results of the knock-out study, emerging lines of evidence suggest that CGRP is still an excellent candidate to be a neuron-derived factor in directing the formation and/or the maintenance of postsynaptic specializations, in particular the regulation of AChE in muscle. Several studies have suggested an AChE-regulating role for CGRP in muscle, including the regulation of AChE T in rodents (14) and chickens (13), the induction of ColQ mRNA (23), the modification of AChE molecular forms (32), and the suppression of PRiMA (Fig. 6) and G 4 AChE (31).…”

“…These results are consistent with the fact that CGRP induced CREB phosphorylation. The CGRP-induced CREB phosphorylation is mediated by a cAMP-dependent signaling pathway, via a CGRP receptor complex on the muscle surface (15,32). In contrast, the mechanism of muscular activity-induced CREB phosphorylation has not yet been determined.…”

Ryanodine‐ and CaMKII‐dependent release of endogenous CGRP induces an increase in acetylcholine quantal size in neuromuscular junctions of mice

“…Of these, CGRP is of special interest because this 37‐amino acid neuropeptide has been found in large dense‐core vesicles (LDCVs) in mammalian motor nerve terminals (Csillik et al., 1993; Matteoli et al., 1988) and is reported to be released as a co‐transmitter in response to sustained depolarization or intense nerve stimulation (Sakaguchi, Inaishi, Kashihara, & Kuno, 1991; Sala, Andreose, Fumagalli, & Lømo, 1995; Uchida et al., 1990) providing the wide spectrum of acute and neurotrophic influences (Buffelli, Pasino, & Cangiano, 2001; Changeux, Duclert, & Sekine, 1992; Correia‐de‐Sá & Ribeiro, 1994; Fernandez, Ross, & Nadelhaft, 1999; Kimura, Okazaki, & Nojima, 1997; Machado et al., 2016; Rossi, Dickerson, & Rotundo, 2003; Salim, Dezaki, Tsuneki, Abdel‐Zaher, & Kimura, 1998). Possible release of endogenous CGRP in response to ryanodine application at resting motor synapses and acute physiological consequences of this release on quantal ACh secretion have not been described yet.…”

Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

“…CGRP is present in motor nerve terminals and is released in an activity-dependent manner (22). Production of cAMP and activation of PKA are induced upon CGRP receptor binding, leading to short-and long-term effects (22): CGRP leads to AChR phosphorylation (23), increased AChR (20,24) and decreased acetylcholine esterase expression (25,26), and NMJ-strength potentiation (27). Thus, CGRP seems to be a trophic factor for the NMJ by local stimulation of cAMP signaling.…”