Calcitonin, amylin, CGRP and adrenomedullin

Overview: Calcitonin (CT), amylin (AMY), calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) receptors (nomenclature as agreed by NC-IUPHAR Subcommittee on CGRP, AM, AMY and CT receptors, see Poyner et al., 2002; Hay et al., 2008) are generated by the genes CALCR (which codes for the CT receptor, ENSG00000064989) and CALCRL (which codes for the CT receptor-like receptor, CL receptor, previously known as CRLR, ENSG00000004948), whose function and pharmacology are altered in the presence of RAMPs (receptor activity-modifying protein). RAMPs are single TM domain proteins of ca 130 amino-acid, identified as a family of three members: RAMP1 (ENSG00000132329), RAMP2 (ENSG00000131477) and RAMP3 (ENSG00000122679). There are splice variants of the CT receptor; these in turn produce variants of the AMY receptor (see Poyner et al., 2002). The endogenous agonists are the peptides CT, αCGRP (also known as CGRP-I), βCGRP (also known as CGRP-II), AMY (also known as islet-amyloid polypeptide, diabetes-associated polypeptide) and AM. There are species differences in peptide sequences, particularly for the CTs. AM2/Intermedin (AM2/IMD) can also activate CGRP, AM₁, AM₂ and AMY₁ receptors, albeit less potently than the cognate agonists (Ogoshi et al., 2003; Roh et al., 2004; Hay et al., 2005). CT receptor-stimulating peptide is another member of the family with selectivity for the CT receptor; it has not been found in humans (Katafuchi et al., 2003). BIBN4096BS is the most selective antagonist available, having a high selectivity for CGRP receptors, with a particular preference for those of primate origin. CGRP-(8-37) acts as an antagonist of CGRP (pK_i 6.5–8.0) and inhibits some AM and AMY responses (7.0). It is inactive at CT receptors. Salmon calcitonin-(8-32) is an antagonist at both AMY and CT receptors. AC187, a salmon CT analogue, is also an antagonist at AMY and CT receptors. Human AM-(22-52) has some selectivity towards AM receptors, but with modest potency, limiting its use.

When co-expressed with RAMP2, the CL receptor produces an AM receptor (AM₁). RAMP3 interacts with the CL receptor to give another receptor that is responsive to AM (AM₂, Fraser et al., 1999). There is some evidence that these AM receptors are pharmacologically distinct (Hay et al., 2003).

Nomenclature	CGRP	AM1	AM2
Composition	CALCRL+RAMP1	CALCRL+RAMP2	CALCRL+RAMP3
Principal transduction	Gs/Gq	Gs	Gs
Rank order of potency	CGRP > AM ≥ AM2/IMD > AMY ≥ salmon CT	AM >> CGRP, AM2/IMD > AMY > salmon CT	AM ≥ CGRP, AM2/IMD > AMY > salmon CT
Selective agonists	αCGRP	AM	AM
Selective antagonists	BIBN4096BS (11, Doods et al., 2000; Hay et al., 2003; 2006;), MK0974 (8.9, Salvatore et al., 2008)	AM-(22–52) (7, Hay et al., 2003)	–
Probes	[125I]-αCGRP (0.1 nM)	[125I]-AM (rat, 0.1–1.0 nM)	[125I]-AM (rat, 0.1–1.0 nM)

Transfection of hCT_(a) with any RAMP can give a receptor with a high affinity for both salmon CT and AMY and varying affinity for different antagonists (Christopoulos et al., 1999; Hay et al., 2005; 2006;). hCT_(a)–RAMP1 [i.e. the AMY_1(a) receptor] has a high affinity for CGRP, unlike hCT_(a)–RAMP3 [i.e. AMY_3(a) receptor] (Christopoulos et al., 1999; Hay et al., 2005). However, AMY receptor phenotype is RAMP-type- and cell-line-dependent (Tilakaratne et al., 2000).

Nomenclature	Calcitonin (CT)	AMY1	AMY2	AMY3
Composition	CALCR	CALCR+RAMP1	CALCR+RAMP2	CALCR+RAMP3
Principal transduction	Gs/Gq	Gs	Gs	Gs
Rank order of potency	Salmon CT ≥ human CT ≥ AMY, CGRP > AM, AM2/IMD	AMY1(a): Salmon CT ≥ AMY ≥ CGRP > AM2/IMD > human CT > AM	Poorly defined	AMY3(a): Salmon CT ≥ AMY > CGRP > AM2/IMD > human CT > AM
Selective agonists	Human CT	AMY	AMY	AMY
Probes	[125I]-CT (salmon, 0.1 nM), [125I]-CT (human, 0.1–1.0 nM)	[125I]-BH-AMY (rat, 0.1–1.0 nM)	[125I]-BH-AMY (rat, 0.1–1.0 nM)	[125I]-BH-AMY (rat, 0.1–1.0 nM)

The agonists described represent the best available, but their selectivity is limited. AM has appreciable affinity for CGRP receptors and some of its effects can be antagonized by CGRP-(8-37). CGRP can show significant cross-reactivity at AMY receptors and some AM receptors. Responsiveness to human CT can be affected by splice variation (at the rat C1b receptor it is very weak, Houssami et al., 1994). Particularly for AMY receptors, relative potency can vary with the type and level of RAMP present and can be influenced by other factors such as G proteins (Tilakaratne et al., 2000). The major splice variant of the calcitonin receptor, CT_(a) has been used for defining the pharmacology of AMY receptors, hence AMY_1(a), etc. (see Poyner et al., 2002 for a full description).

G_s is a prominent route for effector coupling but other pathways (e.g. Ca²⁺ and nitric oxide), and G proteins can be activated. The coupling can be affected by splice variants of the CT receptor [e.g. the 490 amino-acid form of the human receptor, CT_(b), does not cause an increase in intracellular Ca²⁺ and might have low efficacy in generating cAMP]. There is evidence that CGRP-RCP (a 148 amino-acid hydrophilic protein, ENSG00000126522) is important for the coupling of the CL receptor to adenylyl cyclase (Evans et al., 2000).

[¹²⁵I]-Salmon calcitonin is the most common radioligand for CT receptors but it has high affinity for AMY receptors and is also poorly reversible. [¹²⁵I]-Tyr⁰-CGRP is widely used as a radioligand for CGRP receptors.

CGRP₁ and CGRP₂ subtypes have been proposed on the basis of the action of the agonists [Cys(ACM)^2,7]CGRP or [Cys(Et)^2,7]CGRP (putative CGRP₂-selective agents) and antagonist CGRP-(8-37) (CGRP₁-selective, pK_i 7.0–8.0, Juaneda et al., 2000). CALCL/RAMP1 represents the CGRP₁ subtype, previously described in native tissues and cell lines (Aiyar et al., 1996; McLatchie et al., 1998), which is now known simply as the CGRP receptor (Hay et al., 2008). The CGRP₂ receptor is now considered to have arisen from the actions of CGRP at AM₂ and AMY receptors. It is recommended that this term is no longer used (Hay et al., 2008).

Glossary

Abbreviations:

[Cys(ACM)^2,7]CGRP

[acetamidomethyl-Cys^2,7]CGRP

[Cys(Et)^2,7]CGRP

[ethylamide-Cys^2,7]CGRP

AC187

acetyl-[Asn³⁰,Tyr³²]salmon CT

BIBN4096BS

1-piperidinecarboxamide, N-(2-[{5-amino-1-([4-{4-pyridinyl}-1-piperazinyl]carbonyl)pentyl}amino]-1-[{3,5-dibromo-4-hydroxyphenyl}methyl]-2-oxoethyl)-4-(1,4-dihydro-2-oxo-3[2H]-quinazolinyl)

MK0974

N-[(3r,6s)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide

Further Reading

Benemei S, Nicoletti P, Capone JG, Geppetti P (2009). CGRP receptors in the control of pain and inflammation. Curr Opin Pharmacol9: 9–14.

Brain SD, Cox HM (2006). Neuropeptides and their receptors: innovative science providing novel therapeutic targets. Br J Pharmacol147: S202–S211.

Garcia MA, Martin-Santamaria S, de Pascual-Teresa B, Ramos A, Julian M, Martinez A (2006). Adrenomedullin: a new and promising target for drug discovery. Expert Opin Ther Targets10: 303–317.

Gibbons C, Dackor R, Dunworth W, Fritz-Six K, Caron KM (2007). Receptor activity-modifying proteins: RAMPing up adrenomedullin signaling. Mol Endocrinol21: 783–796.

Hay DL, Poyner DR, Sexton PM (2006). GPCR modulation by RAMPs. Pharmacol Ther109: 173–197.

Hay DL, Poyner DR, Quirion R (2008). International Union of Pharmacology. LXIX. Status of the calcitonin gene-related peptide subtype 2 receptor. Pharmacol Rev60: 143–145.

Ishimitsu T, Ono H, Minami J, Matsuoka H (2006). Pathophysiologic and therapeutic implications of adrenomedullin in cardiovascular disorders. Pharmacol Ther111: 909–927.

Nikitenko LL, Fox SB, Kehoe S, Rees MC, Bicknell R (2006). Adrenomedullin and tumour angiogenesis. Br J Cancer94: 1–7.

Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W et al. (2002). International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev54: 233–246.

Recober A, Russo AF (2009). Calcitonin gene-related peptide: an update on the biology. Curr Opin Neurol22: 241–246.