Calcitonin gene-related peptide (CGRP) receptors are linked to cyclic adenosine monophosphate production in SK-N-MC human neuroblastoma cells

doi:10.1016/0304-3940(90)90832-T

Neuroscience Letters

Volume 119, Issue 2, 13 November 1990, Pages 195-198

https://doi.org/10.1016/0304-3940(90)90832-T Get rights and content

Abstract

Calcitonin gene-related peptide (CGRP) stimulated cyclic adenosine monophosphate (cAMP) levels in SK-N-MC human neuroblastoma cells in a time- and concentration-dependent manner. The efficacy order for CGRPs was human α-CGRP = human β-CGRP = chick CGRP > rat CGRP > human [Tyr⁰]CGRP. Calcitonin (CT) failed to influence cAMP production in SK-N-MC cells. [Tyr⁰]CGRP_28–37 which by itself did not affect cAMP levels antagonized CGRP action. Saturation analysis using [¹²⁵I]CGRP showed a homogeneous population of binding sites. CGRP but not CT, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) inhibited radioligand binding. Our results provide evidence that human neuroblastoma SK-N-MC cells contain highly specific CGRP receptors which are positively coupled to cAMP generation.

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Receptor component protein, an endogenous allosteric modulator of family B G protein coupled receptors
2020, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
For the AM1 receptor, GIPR and GLP1R, there were no significant changes to Emax or pEC50. We next investigated whether RCP knock down affected the signalling of endogenously expressed family B receptors in SK-N-MC [16], HEK293T [12] and HUVEC [17] cells. RCP knockdown by siRNA reduces agonist stimulated cAMP production by endogenously expressed CGRPR in SK-N-MC cells (Fig. 4a).
Receptor component protein (RCP) is a 148 amino acid intracellular peripheral membrane protein, previously identified as promoting the coupling of CGRP to cAMP production at the CGRP receptor, a heterodimer of calcitonin receptor like-receptor (CLR), a family B G protein-coupled receptor (GPCR) and receptor activity modifying protein 1 (RAMP1). We extend these observations to show that it selectively enhances CGRP receptor coupling to Gs but not Gq or pERK activation. At other family B GPCRs, it enhances cAMP production at the calcitonin, corticotrophin releasing factor type 1a and glucagon-like peptide type 2 receptors with their cognate ligands but not at the adrenomedullin type 1 (AM₁), gastric inhibitory peptide and glucagon-like peptide type 1 receptors, all expressed in transfected HEK293S cells. However, there is also cell-line variability as RCP did not enhance cAMP production at the endogenous calcitonin receptor in HEK293T cells and it has previously been reported that it is active on the AM₁ receptor expressed on NIH3T3 cells. RCP appears to behave as a positive allosteric modulator at coupling a number of family B GPCRs to Gs, albeit in a manner that is regulated by cell-specific factors. It may exert its effects at the interface between the 2nd intracellular loop of the GPCR and Gs, although there is likely to be some overlap between this location and that occupied by the C-terminus of RAMPs if they bind to the GPCRs.
Activation of the cation channel TRPM3 in perivascular nerves induces vasodilation of resistance arteries
2019, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
Next, we explored the mechanisms by which CGRP receptor activation in VSMC leads to vasodilation. Stimulation of the CGRP receptor has been reported to increase cyclic adenosine monophosphate (cAMP) production via the adenylate cyclase (AC) [26] and may therefore lead to protein kinase A (PKA)-mediated activation of K+ channels in VSMC and consequent hyperpolarization and arterial relaxation. To test whether activation of K+ channels is involved in the vasodilation response triggered by TRPM3 activation we compared the effects of 10 μM PS in arteries treated or not with K+ channel blockers.
The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca²⁺-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC₅₀, of 14 and 100 μM). The low EC₅₀ component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K⁺ channel blockers, and not mediated by β-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K⁺ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries.
Calcitonin gene-related peptide mediates an inflammatory response in Schwann cells via cAMP-dependent ERK signaling cascade
2016, Life Sciences
Citation Excerpt :
CGRP receptor component protein has been shown to be required for G-protein-coupled signal transduction at receptors activated by CGRP [16,31]. Although the second messenger system of CGRP receptor stimulation has been characterized quite well to involve generation of cAMP through coupled with G-protein (Gs), the distal signaling components beyond the second messenger generation are not currently well understood. [40] showed that this ERK activation by CGRP acts through Gs coupled receptors in SK-N-MC cells [40].
Calcitonin gene-related peptides (CGRP), an endogenous neuropeptide, play an important role in the development of neuroinflammation by acting upon its receptor. The CGRP receptor immunoreactivity was identified on Schwann cells. However the effects of CGRP on Schwann cells are unknown and the exact signaling mechanisms associated with CGRP receptor activation related to Schwann cells inflammatory responses are not well understood. We investigated the effect of CGRP on CGRP receptor activation mediates a proinflammatory signaling response in Schwann cells.
CGRP-induced ERK-MAPK phosphorylation and proinflammatory cytokines, interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) expressions were measured by immune blotting. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including CGRP (8–37), SQ 22536 and H-89.
Treatment with CGRP demonstrated a significant generation of IL-1β and IL-6 but not in the level of TNF-α. In addition, there was a temporal increase in the activated form of ERK caused by CGRP that was prevented after pretreatment with CGRP (8–37), SQ 22536 and H-89. Furthermore, use of the CGRP (8–37), ERK inhibitor PD 98059, SQ 22536 or H-89 abolished the CGRP mediated increase in IL-1β.
This investigation provides evidence for a novel CGRP activation on Schwann cells that mediates inflammatory response by increasing of IL-1β and IL-6 expression. CGRP activates the cAMP-PKA-ERK signaling cascade leading to IL-1β production. These results support the notion that CGRP may play a direct role to initiate inflammatory processes in the peripheral nervous system.
Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract
2012, Peptides
Citation Excerpt :
Images of stained and control slides were collected and processed (colored and merged) identically. To determine if mRNA for CLR and RAMP1 were expressed in the normal human GI tract, we isolated RNA from stomach, ileum and colon biopsies/segments and from SK-N-MC cells (known to express CLR and RAMP1 [37]). Negative control without RT enzyme did not yield a PCR product, whereas, CLR and RAMP1 products were amplified from the human GI segments and SK-N-MC cells (Fig. 1).
Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.
Localization of the Calcitonin Gene-related Peptide Receptor Complex at the Vertebrate Neuromuscular Junction and Its Role in Regulating Acetylcholinesterase Expression
2003, Journal of Biological Chemistry
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 CGRP-binding 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 CGRP-binding protein referred to as the calcitonin receptor-like 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.
Molecular cloning and characterization of mouse calcitonin gene-related peptide receptor
2002, Neuropeptides
The calcitonin gene-related peptide (CGRP) plays important roles as a neurotransmitter/neuromodulator in the central nervous system, and as a potent vasodilator when secreted from peripheral, perivascular nerves through its specific receptors. In this study, we cloned mouse cDNA counterparts of the human CGRP receptor composed of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) and examined the signal transduction mechanism through the CGRP receptor. Mouse CRLR (mCRLR) is a 462-amino acid G protein-coupled heptahelical receptor, and mouse RAMP1 (mRAMP1) is a 148-amino acid single membrane-spanning protein with a short cytoplasmic portion. Specific binding of ¹²⁵I-CGRP was detected only when both mCRLR and mRAMP1 cDNAs were cotransfected to COS-7 cells, and the Kd value of the receptor was 2.2×10⁻¹⁰ M. CGRP induced a marked elevation of the intracellular cAMP levels in COS-7 cells cotransfected with mCRLR and mRAMP1. CGRP signaling through the mCRLR/mRAMP1 receptor complex was found to increase the promoter activities of cyclic AMP responsive element and serum responsive element in the co-transfected HeLa cells. These results indicate that mCRLR and mRAMP1 constitute a functional mouse CGRP receptor for the transduction of CGRP signaling by PKA and extracellular signal-regulated kinase signal transduction pathways.

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Calcitonin gene-related peptide (CGRP) receptors are linked to cyclic adenosine monophosphate production in SK-N-MC human neuroblastoma cells

Abstract

Biochem. Biophys. Res. Commun.

Neurosci. Lett.

Neurochem. Int.

Bone

FEBS Lett.

Biochem. Biophys. Res. Commun.