Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23–60 of the calcitonin receptor-like receptor

Taddese

et al. 2013

J. R. Soc. Interface.

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The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.

Section: Resultsmentioning

confidence: 99%

The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

Woolley

Taddese

et al. 2013

J. R. Soc. Interface.

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“…The effects of Y49 are particularly notable; mutation of this residue abolishes binding and substantially reduces expression in the CGRP receptor (Barwell et al ., 2010). Similar effects on cell surface expression are seen in the AM 1 receptor.…”

Section: Discussionmentioning

confidence: 99%

Receptor activity‐modifying protein‐dependent effects of mutations in the calcitonin receptor‐like receptor: implications for adrenomedullin and calcitonin gene‐related peptide pharmacology

Walker

et al. 2014

British J Pharmacology

Background and PurposeReceptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear.Experimental ApproachGuided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants.Key ResultsAn important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide.Conclusions and ImplicationsRAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2.

“…This is illustrated in Figure 3A. In the N‐terminal α‐helix of CLR, the residue I41 was demonstrated to be important for CGRP binding (Barwell et al ., 2010) (Table 1, Figure 3A). According to our model shown in Figure 3B, M48 of CTR is found in the equivalent position to I41 of CLR; it is possible that this residue may also be involved in ligand interactions.…”

Section: The N‐terminal Ecd Is Essential For Peptide Bindingmentioning

confidence: 96%

“…Despite lacking a peptide‐bound ECD structure for CT peptide family receptors, some insights have been gained from mutagenesis and photoaffinity labelling studies on these receptors. Similar to other family B GPCRs, residues within the ECD of CTR and CLR are critical for ligand binding (Dong et al ., 2004a; Banerjee et al ., 2006; Barwell et al ., 2010). The extreme N‐terminus of CLR (residues 23–60) not only contributes to the selectivity of the interactions with either AM or CGRP (Koller et al ., 2002) but is required for RAMP1 association (Ittner et al ., 2005; Barwell et al ., 2010; ter Haar et al ., 2010).…”

Section: The N‐terminal Ecd Is Essential For Peptide Bindingmentioning

confidence: 99%

Calcitonin and calcitonin receptor‐like receptors: common themes with family B GPCRs?

Barwell

Gingell

et al. 2012

British J Pharmacology

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The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR.