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

Abstract

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.

Figure 1

Amino acid sequence alignment of selected family B GPCRs generated using clustal-w and ESPript. (A) Aligned secondary structure of extracellular domains. Secondary structural elements of CLR and the PTH₁ receptor are shown above and below the alignment respectively. The signal peptide region has been omitted. (B) Alignment of the TM helices predicted by TMHMM for CLR (above) and the PTH₁ receptor (below), loops and C-terminus. Conserved regions are shown in solid red boxes and regions of similarity in yellow, cysteines forming disulphide bridges as determined by X-ray structure are indicated by a black triangle. Note that the entire sequences of the receptors were aligned but divided into parts (A) and (B) for the purposes of this figure.

Figure 1

Amino acid sequence alignment of selected family B GPCRs generated using clustal-w and ESPript. (A) Aligned secondary structure of extracellular domains. Secondary structural elements of CLR and the PTH₁ receptor are shown above and below the alignment respectively. The signal peptide region has been omitted. (B) Alignment of the TM helices predicted by TMHMM for CLR (above) and the PTH₁ receptor (below), loops and C-terminus. Conserved regions are shown in solid red boxes and regions of similarity in yellow, cysteines forming disulphide bridges as determined by X-ray structure are indicated by a black triangle. Note that the entire sequences of the receptors were aligned but divided into parts (A) and (B) for the purposes of this figure.

Figure 2

Schematic diagram of CLR (purple) with RAMP1 (green) (the CGRP receptor). Like other family B GPCRs, CLR is divided into functional domains: the N-terminal (NT) extracellular domain (ECD) is important for peptide binding; the extracellular loops (ECL) and upper transmembrane (TM) domain are collectively known as the juxtamembrane (JM) domain and are involved in peptide binding and receptor activation; the TM domain undergoes a conformational change upon activation and the intracellular loops (ICL) and receptor C-terminus (CT) are involved in interactions with intracellular proteins such as G proteins and β-arrestin. Amino acid residues are numbered from the start of the predicted N-terminal signal peptide. Some important residues, which have been discussed in the text or Table 1 are highlighted: I41 and N123 in the ECD, R173 in ICL1, P343 and R336 in TM6 and W399 in helix 8 of the C-terminus. The boxed region of the CLR C-terminus has been reported to be involved in receptor internalization. Helices in CLR and RAMP1 are represented as cylinders.

Figure 3

RAMP1 interaction with CLR and CTR via their ECDs. (A) CLR and RAMP1 X-ray crystal structure (Ter Haar et al., 2010) and, (B) Homology model of CTR and RAMP1, generated using (A). CLR is in purple, RAMP1 in green and CTR in orange. Selected residues in the N-terminal α-helix of CLR, which are important for peptide (I41) or RAMP1 interactions are shown in stick format. The equivalent residues, which may share similar roles, are shown in CTR.

Figure 4

The calcitonin family of peptides contain a conserved disulfide that mimics the helix N-cap motif. (A) N-terminal region of a human calcitonin analogue elucidated by solution NMR in the presence of sodium dodecyl micelles (Andreotti et al., 2011; PDB 2JXZ). The peptide is depicted as a red ribbon and amino acids are labelled with single letter identification. The conserved disulfide bond and side chains of cysteine-1 and cysteine-7 are highlighted yellow. Asparagine-3 side chain is highlighted (blue) as it corresponds to the N' residue as defined by Neumann et al. (2008). (B) Receptor-bound N-terminal region of PACAP(1–21)NH₂ (Inooka et al., 2001). The peptide is depicted as a blue ribbon. Neumann et al. (2008) defined PACAP as having a simple IA type helix N-cap motif. Phenylalanine-6 (highlighted green) represents the N' residue. Threonine-7 (highlighted red) represents the N-cap residue. Tyrosine-10 (highlighted purple) represents N3 residue. (C) A multiple sequence alignment of the helix N-cap regions of the calcitonin family of ligands [calcitonin, αCGRP, βCGRP, amylin, AM and AM-2 (also referred to as intermedin)] and PACAP. The calcitonin family peptides have been aligned using ClustalW and then aligned to PACAP based on the helix N-cap motif. Residues highlighted red in the PACAP sequence represent the N', N-cap and N3 residues respectively. Residues highlighted red in the calcitonin family of ligands represent the conserved cysteine residues that participate in the disulfide bond and the equivalent N' residue.

Figure 5

A speculative helical wheel representation of the CLR transmembrane bundle as viewed from the extracellular surface to show the analogous major and minor binding pockets found in family A GPCRs. Periodicity of each TM domain was approximated based on the Vohra et al. (2007) strategy coupled with a refinement procedure used to investigate speculative models of CLR based on bovine rhodopsin.