Effects of two truncated forms of human calcitonin-gene related peptide: implications for receptor classification

doi:10.1016/0014-2999(94)90222-4

European Journal of Pharmacology

Volume 265, Issues 1–2, 14 November 1994, Pages 53-59

https://doi.org/10.1016/0014-2999(94)90222-4 Get rights and content

Abstract

We investigated the possibility that human α-calcitonin-gene related peptide (CGRP)-(8–37) and human βCGRP-(8–37) show some selectivity as antagonists of CGRP₁ and CGRP₂ receptor-mediated responses. Bindings assays showed that human αCGRP, human αCGRP-(8–37) and human βCGRP-(8–37) showed high affinity (in the nanomolar concentration range) for CGRP receptors expressed in SK-N-MC cells and also in rat brain membrain preparations. Both human αCGRP-(8–37) and human βCGRP-(8–37) were potent antagonists of human αCGRP-stimulated cAMP accumulation in SK-N-MC cells. However, both human αCGRP-(8–37) and human βCGRP-(8–37) were weakly effective in antagonizing human αCGRP-stimulated responses in guinea-pig atria and rat vas deferens. In rat vas deferens, but not guinea-pig atria, the effects of human αCGRP and human αCGRP-(8–37) (but not human βCGRP-(8–37)) were potentiated by thirophan. Neither human α- nor human βCGRP-(8–37) showed selectivity for supposedly CGRP₁ and CGRP₂ receptor-mediated responses. Furthermore, differences in the effects of the truncated CGRP analogues may reflect differences in enzyme distribution rather than the existence of CGRP receptor subtypes.

References (17)

I. Jansen
Characterisation of calcitonin gene-related peptide (CGRP) receptors in guinea-pig basilar artery
Neuropeptides
(1992)
M. Katayama et al.
Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase
Peptides
(1991)
P. Le Greves et al.
Calcitonin gene-related peptide is metabolized by an endopeptidase hydrolyzing substance P
Regul. Pept.
(1989)
H. Nakamuta et al.
Binding sites of calcitonin gene-related peptide (CGRP). Abundant occurrence in visceral organs
Jpn. J. Pharmacol.
(1986)
Y. Okimura et al.
Calcitonin gene-related peptide-like immunoreactivity in the central nervous system and peripheral organs of the rat
Regul. Pept.
(1987)
G. Skofitsch et al.
Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system
Peptides
(1985)
I.J.M. Beresford et al.
Investigation into species variation in tachykinin NK₁-receptors by use of the non-peptide antagonist CP-96,345
Br. J. Pharmacol.
(1991)
L.H. Breimer et al.
Peptides from the calcitonin genes: molecular genetics, structure and function
Biochem. J.
(1988)

There are more references available in the full text version of this article.

Cited by (44)

Characterization of capsaicin induced responses in mice vas deferens: Evidence of CGRP uptake
2011, European Journal of Pharmacology
Citation Excerpt :
These functional studies have revealed the presence of both TRPV1 receptors and CGRP receptors (Calcrl + RAMP1) in vas deferens. Earlier studies report different functional potencies of CGRP and its peptide family in various tissues (Dennis et al., 1990; Dumont et al., 1997; Longmore et al., 1994; Wisskirchen et al., 1998), which may partially be attributed to the relative expression levels of receptor activity modifying protein (RAMPs 1–3) as well as the difference in receptor density. Analysis of the effects induced by capsaicin or by an individual endogenous peptide is often hampered by the co-release of multiple neuropeptides from sensory nerve endings in many tissues.
Calcitonin gene-related peptide (CGRP) is extensively distributed in primary afferent sensory nerves, including those innervating the genitourinary tract. Capsaicin can stimulate the release of CGRP from intracellular stores of these nerves, but this phenomenon has not been investigated in-depth in isolated preparations. The present study sets out to study and characterize the capsaicin as well as CGRP-induced responses in isolated mouse vas deferens. The effects of capsaicin and CGRP family of peptides were studied on electrically-induced twitch responses in the absence or presence of transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1) antagonist and CGRP receptor antagonists. Twitch responses were attenuated by capsaicin (1 nM–30 nM) and CGRP family of peptides. The potency order was CGRP > intermedin-long (IMDL) ~ [Cys(Et)^2,7]αCGRP ~ adrenomedullin (AM) > [Cys(ACM)^2,7]αCGRP > amylin (AMY). These responses were disinhibited by the CGRP receptor antagonists and TRPV1 antagonists. The addition of CGRP receptor antagonists caused a transient potentiation of the twitch response and this potentiation was blocked by pretreatment with capsaicin and enhanced by incubation with exogenous CGRP. During the second consecutive cumulative concentration-response curve with capsaicin, the first phase of concentration-response curve disappeared and this was partially restored when the mouse vas deferens was preincubated with CGRP, suggesting the uptake of exogenous CGRP by nerves. Besides showing capsaicin-induced CGRP releases this study shows that exogenous CGRP can be taken up in vas deferens and can be re-released. CGRP uptake will add another dimension in understanding the homeostasis of this neuropeptide.
Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1
2007, Journal of Biological Chemistry
Citation Excerpt :
These results show that ubiquitination is not required for the post-endocytic sorting of CLR or RAMP1 to lysosomes for degradation. CLR and RAMP1 Can Recycle and Traffic to Lysosomes in SK-N-MC Cells—To determine whether CGRP can induce recycling and lysosomal trafficking of CLR and RAMP1 in cells naturally expressing these proteins, we studied human SK-N-MC cells (28). The CLR antibody RK11 is raised to the C-terminal 18 residues of rat CLR, which differs from human CLR by 4 amino acid substitutions and thus does not fully cross-react with the human receptor.3
Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10^-7 m CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca²⁺]_i. Cycloheximide did not affect resensitization, but bafilomycin A₁, an inhibitor of vacuolar H⁺-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10^-7 m CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded ∼4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.
Cys<sup>2,7</sup>EtαCGRP is a potent agonist for CGRP<inf>1</inf> receptors in SK-N-MC cells
2005, Biochemical Pharmacology
The present study reveals that cystein^2,7 ethyl-amideαCGRP (Cys^2,7EtαCGRP), an advertised calcitonin gene-related peptide 2 (CGRP₂) receptor subtype-selective agonist, is also a potent agonist for the calcitonin gene-related peptide 1 (CGRP₁) receptors natively expressed in the SK-N-MC human neuroblastoma cell line. Cys^2,7EtαCGRP and α calcitonin gene-related peptide (αCGRP) promote cyclic AMP accumulation in intact SK-N-MC cells to the same extent with EC₅₀ of 1.6 ± 0.2 and 0.4 ± 0.08 nM, respectively. The antagonist α calcitonin gene-related peptide-8-37 (αCGRP-(8-37)) produces a concentration-dependent rightward shift of the αCGRP- and Cys^2,7EtαCGRP concentration–response curves with K_B-values (71 ± 33 and 47 ± 21 nM, respectively). The competitive antagonism by αCGRP-(8-37) and the similar K_B-values suggests that αCGRP and Cys^2,7EtαCGRP stimulate the same receptor. In competition binding studies with [¹²⁵I]-αCGRP on SK-N-MC cell membranes, Cys^2,7EtαCGRP and αCGRP-(8-37) display high affinity for the majority of the binding sites with K_i-values of 0.030 ± 0.013 and 0.60 ± 0.013 nM, respectively. The present findings are at odds with the proclaimed utilization of Cys^2,7EtαCGRP as a CGRP₂ receptor-selective pharmacological tool. Differences between the agonistic profile of this ligand in this and other experimental systems might be species – or even cell type – dependent.
The pharmacology of CGRP-responsive receptors in cultured and transfected cells
2004, Peptides
Historically, CGRP receptors have been classified as CGRP₁ or CGRP₂ subtypes, chiefly depending on their affinity for the antagonist CGRP_8–37. It has been shown that the complex between calcitonin receptor-like receptor (CRLR or CL) and receptor activity modifying protein (RAMP) 1 provides a molecular correlate for the CGRP₁ receptor; however, this does not explain the range of affinities seen for CGRP_8–37 in isolated tissues. It is suggested that these may largely be explained by a combination of methodological factors and CGRP-responsive receptors generated by CL and RAMP2 or RAMP3 and complexes of RAMPs with the calcitonin receptor.
Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists
2002, Journal of Biological Chemistry
Citation Excerpt :
These results demonstrated that RAMP1 determined the affinity of BIBN4096BS and Compound 1 for human and rat CGRP receptors. By contrast, the peptide antagonist CGRP8–37was not species-selective, resulting in Ki values of 1.3 and 1.2 nm for the human and rat receptors, respectively (data not shown), consistent with previous reports (2,21). The Kd of the radioligand125I-hCGRP also was similar for both human and rat receptors, as well as for the mixed-species receptors (Supplementary Material Table SI).
The heterodimeric CGRP receptor requires co-expression of calcitonin receptor-like receptor (CRLR) and an accessory protein called receptor activity-modifying protein (RAMP) 1 (McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998) Nature 393, 333–339). Several non-peptide CGRP receptor antagonists have been shown to exhibit marked species selectivity, with >100-fold higher affinities for the human CGRP receptor than for receptors from other species (Doods, H., Hallermayer, G., Wu, D., Entzeroth, M., Rudolf, K., Engel, W., and Eberlein, W. (2000) Br. J. Pharmacol. 129, 420–423; Edvinsson, L., Sams, A., Jansen-Olesen, I., Tajti, J., Kane, S. A., Rutledge, R. Z., Koblan, K. S., Hill, R. G., and Longmore, J. (2001) Eur. J. Pharmacol. 415, 39–44). This observation provided an opportunity to map the determinants of receptor affinity exhibited by BIBN4096BS and the truncated analogs, Compounds 1 and 2. All three compounds exhibited higher affinity for the human receptor, human CRLR/human RAMP1, than for the rat receptor, rat CRLR/rat RAMP1. We have now demonstrated that this species selectivity was directed exclusively by RAMP1. By generating recombinant human/rat CRLR/RAMP1 receptors, we demonstrated that co-expression of human CRLR with rat RAMP1 produced rat receptor pharmacology, and vice versa. Moreover, with rat/human RAMP1 chimeras and site-directed mutants, we have identified a single amino acid at position 74 of RAMP1 that modulates the affinity of small molecule antagonists for CRLR/RAMP1. Replacement of lysine 74 in rat RAMP1 with tryptophan (the homologous amino acid in the human receptor) resulted in a ≥100-fold increase in antagonist affinities, similar to theK_i values for the human receptor. These observations suggest that important determinants of small molecule antagonist affinity for the CGRP receptor reside within the extracellular region of RAMP1 and provide evidence that this receptor accessory protein may participate in antagonist binding.
Characterization of calcitonin gene-related peptide (CGRP) receptors and their receptor-activity-modifying proteins (RAMPs) in human brain microvascular and astroglial cells in culture
2002, Neuropharmacology
- 1.
  In the present study, we examined the expression of the CGRP receptor-activity-modifying proteins (RAMP1, RAMP2 and RAMP3) and receptor component protein (RCP) in human brain astrocytes (AST), cerebromicrovascular endothelial (EC) and smooth muscle (SMC) cells in culture. Further, we pharmacologically characterized CGRP receptors in these cells by assessing the potency of the CGRP receptor antagonists h-αCGRP_8-37 and the new non-peptide compound BIBN4096BS to block the production of cAMP elicited by CGRP₁ and CGRP₂ receptor agonists.
- 2.
  AST, EC and SMC all expressed mRNAs for RAMP1, RAMP2 and RCP. In contrast, message for RAMP3 was detected in AST, but not in SMC and in only one out of four preparations of EC.
- 3.
  h-αCGRP, h-βCGRP and [Cys (Et)^2,7]-h-αCGRP exerted concentration-dependent production of cAMP in all cultures, with a maximal effect at 25–50 nM (20–60-fold increase from basal levels). In contrast, 50 nM [Cys (Acm)^2,7]-h-αCGRP only induced a weak stimulatory effect on cAMP formation, especially in SMC and AST (1.5- and 5-fold increase above baseline, respectively).
- 4.
  h-αCGRP_8-37 and BIBN4096BS concentration-dependently inhibited cAMP formation evoked by CGRP receptor agonists. Depending on the agonists used, h-αCGRP_8-37 distinguished two different CGRP receptors for which it exhibited low (pIC₅₀≤6.4) and high (pIC₅₀∼7.3) affinity, respectively. BIBN4096BS was much more potent (>2.5 orders of magnitude) than h-αCGRP_8-37. Further, BIBN4096BS was able to discriminate three different CGRP receptor sites for which it exhibited low (pIC₅₀∼9.3–9.9), intermediate (pIC₅₀∼10.9), and a very high (pIC₅₀∼13.7) affinity, respectively. Together, these results suggest the presence of CGRP₁ and/or CGRP₂ receptors in human brain AST, EC and SMC, and of an additional population of CGRP receptors in AST, possibly associated to the combined expression of RAMP3 and RCP in these cells, for which BIBN4096BS exhibits an exquisitely high affinity.

View all citing articles on Scopus

View full text

Effects of two truncated forms of human calcitonin-gene related peptide: implications for receptor classification

Abstract

Neuropeptides

Peptides

Regul. Pept.

Jpn. J. Pharmacol.

Regul. Pept.

Peptides

Investigation into species variation in tachykinin NK1-receptors by use of the non-peptide antagonist CP-96,345

Br. J. Pharmacol.

Peptides from the calcitonin genes: molecular genetics, structure and function

Biochem. J.

Investigation into species variation in tachykinin NK₁-receptors by use of the non-peptide antagonist CP-96,345