ObjectiveThe trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system.MethodsReceptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons.ResultsmRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons.InterpretationThe unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine.

The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogues of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated C-terminus and receptor "capture" and "activation" regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide subregions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics, and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide−receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity.

Background Calcitonin gene-related peptide is an important target for migraine and other painful neurovascular conditions. Understanding the normal biological functions of calcitonin gene-related peptide is critical to understand the mechanisms of calcitonin gene-related peptide-blocking therapies as well as engineering improvements to these medications. Calcitonin gene-related peptide is closely related to other peptides in the calcitonin gene-related peptide family of peptides, including amylin. Relatedness in peptide sequence and in receptor biology makes it difficult to tease apart the contributions that each peptide and receptor makes to physiological processes and to disorders. Summary The focus of this review is the expression of calcitonin gene-related peptide, related peptides and their receptors in the central nervous system. Calcitonin gene-related peptide is expressed throughout the nervous system, whereas amylin and adrenomedullin have only limited expression at discrete sites in the brain. The components of two receptors that respond to calcitonin gene-related peptide, the calcitonin gene-related peptide receptor (calcitonin receptor-like receptor with receptor activity-modifying protein 1) and the AMY receptor (calcitonin receptor with receptor activity-modifying protein 1), are expressed throughout the nervous system. Understanding expression of the peptides and their receptors lays the foundation for more deeply understanding their physiology, pathophysiology and therapeutic use.

Amylin is an important, but poorly understood, 37 amino acid glucoregulatory hormone with great potential to target metabolic diseases. A working example that the amylin system is one worth developing is the FDA-approved drug used in insulin-requiring diabetic patients, pramlintide. However, certain characteristics of pramlintide pharmacokinetics and formulation leave considerable room for further development of amylin-mimetic compounds. Given that amylin-mimetic drug design and development is an active area of research, surprisingly little is known about the structure/function relationships of amylin. This is largely due to the unfavourable aggregative and solubility properties of the native peptide sequence, which are further complicated by the composition of amylin receptors. These are complexes of the calcitonin receptor with receptor activity-modifying proteins. This review explores what is known of the structure-function relationships of amylin and provides insights that can be drawn from the closely related peptide, CGRP. We also describe how this information is aiding the development of more potent and stable amylin mimetics, including peptide hybrids.

Islet amyloid formation contributes to β-cell death and dysfunction in type-2 diabetes and to the failure of islet transplants. Amylin (islet amyloid polypeptide, IAPP), a normally soluble 37 residue polypeptide hormone produced in the pancreatic β-cells, is responsible for amyloid formation in type-2 diabetes and is deficient in type-1 diabetes. Amylin normally plays an adaptive role in metabolism, and the development of nontoxic, non-amyloidogenic, bioactive variants of human amylin are of interest for use as adjuncts to insulin therapy. Naturally occurring non-amyloidogenic variants are of interest for xenobiotic transplantation and because they can provide clues toward understanding the amyloidogenicity of human amylin. The sequence of amylin is well-conserved among species, but sequence differences strongly correlate with in vitro amyloidogenicity and with islet amyloid formation in vivo. Bovine amylin differs from the human peptide at 10 positions and is one of the most divergent among known amylin sequences. We show that bovine amylin oligomerizes but is not toxic to cultured β-cells and that it is considerably less amyloidogenic than the human polypeptide and is only a low-potency agonist at human amylin-responsive receptors. The bovine sequence contains several nonconservative substitutions relative to human amylin, including His to Pro, Ser to Pro, and Asn to Lys replacements. The effect of these substitutions is analyzed in the context of wild-type human amylin; the results provide insight into their role in receptor activation, the mode of assembly of human amylin, and the design of soluble amylin analogues.

Background and Purpose: The calcitonin (CT) receptor family is complex, comprising two receptors (the CT receptor [CTR] and the CTR-like receptor [CLR]), three accessory proteins (RAMPs) and multiple endogenous peptides. This family contains several important drug targets, including CGRP, which is targeted by migraine therapeutics. The pharmacology of this receptor family is poorly characterised in species other than rats and humans. To facilitate understanding of translational and preclinical data, we need to know the receptor pharmacology of this family in mice.Experimental Approach: Plasmids encoding mouse CLR/CTR and RAMPs were transiently transfected into Cos-7 cells. cAMP production was measured in response to agonists in the absence or presence of antagonists. Key Results:We report the first synthesis and characterisation of mouse adrenomedullin, adrenomedullin 2 and βCGRP and of mouse CTR without or with mouse RAMPs. Receptors containing m-CTR had subtly different pharmacology than human receptors; they were promiscuous in their pharmacology, both with and without RAMPs. Several peptides, including mouse αCGRP and mouse adrenomedullin 2, were potent agonists of the m-CTR:m-RAMP3 complex. Pharmacological profiles of receptors comprising m-CLR:m-RAMPs were generally similar to those of their human counterparts, albeit with reduced specificity. Conclusion and Implications:Mouse receptor pharmacology differed from that in humans, with mouse receptors displaying reduced discrimination between ligands. This creates challenges for interpreting which receptor may underlie an effect in preclinical models and thus translation of findings from mice to humans. It also highlights the need for new ligands to differentiate between these complexes.

The calcitonin receptor (CTR) is relevant to three hormonal systems: amylin, calcitonin, and calcitonin gene-related peptide (CGRP). Receptors for amylin and calcitonin are targets for treating obesity, diabetes, and bone disorders. CGRP receptors represent a target for pain and migraine. Amylin receptors (AMY) are a heterodimer formed by the coexpression of CTR with receptor activity-modifying proteins (RAMPs). CTR with RAMP1 responds potently to both amylin and CGRP. The brain stem is a major site of action for circulating amylin and is a rich site of CGRP binding. This study aimed to enhance our understanding of these hormone systems by mapping CTR expression in the human brain stem, specifically the medulla oblongata. Widespread CTR-like immunoreactivity was observed throughout the medulla. Dense CTR staining was noted in several discrete nuclei, including the nucleus of the solitary tract, the hypoglossal nucleus, the cuneate nucleus, spinal trigeminal nucleus, the gracile nucleus, and the inferior olivary nucleus. CTR staining was also observed in the area postrema, the lateral reticular nucleus, and the pyramidal tract. The extensive expression of CTR in the medulla suggests that CTR may be involved in a wider range of functions than currently appreciated.

Class B G protein-coupled receptors are highly therapeutically relevant but challenges remain in identifying suitable small-molecule drugs. The calcitonin-like receptor (CLR) in particular is linked to conditions such as migraine, cardiovascular disease, and inflammatory bowel disease. The CLR cannot act as a cell-surface receptor alone but rather must couple to one of three receptor activity-modifying proteins (RAMPs), forming heterodimeric receptors for the peptides adrenomedullin and calcitonin gene-related peptide. These peptides have extended binding sites across their receptors. This is one reason why there are few small-molecule ligands that can modulate these receptors. Here we describe small molecules that are able to positively modulate the signaling of the CLR with all three RAMPs but are not active at the related calcitonin receptor. These compounds were selected from a β-arrestin recruitment screen, coupled with rounds of medicinal chemistry to improve their activity. Translational potential is shown as the compounds can positively modulate cAMP signaling in a vascular cell line model. Binding experiments do not support an extracellular domain binding site; however, molecular modeling reveals potential allosteric binding sites in multiple receptor regions. These are the first small-molecule positive modulators described for the CLR:RAMP complexes.