Functional modulation of PTH1R activation and signalling by RAMP2

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs) including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR, and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signalling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signalling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signalling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R. Employing homology modelling we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signalling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

Calcitonin receptors in GtoPdb v.2021.2

This receptor family comprises a group of receptors for the calcitonin/CGRP family of peptides. The calcitonin (CT), amylin (AMY), calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CGRP, AM, AMY, and CT receptors [131, 74, 71]) are generated by the genes CALCR (which codes for the CT receptor) and CALCRL (which codes for the calcitonin receptor-like receptor, CLR, previously known as CRLR). Their function and pharmacology are altered in the presence of RAMPs (receptor activity-modifying proteins), which are single TM domain proteins of ca. 150 amino acids, identified as a family of three members; RAMP1, RAMP2 and RAMP3. There are splice variants of the CT receptor; these in turn produce variants of the AMY receptor [131], some of which can be potently activated by CGRP. The endogenous agonists are the peptides calcitonin, α-CGRP (formerly known as CGRP-I), β-CGRP (formerly known as CGRP-II), amylin (occasionally called islet-amyloid polypeptide, diabetes-associated polypeptide), adrenomedullin and adrenomedullin 2/intermedin. There are species differences in peptide sequences, particularly for the CTs. CTR-stimulating peptide (CRSP) is another member of the family with selectivity for the CT receptor but it is not expressed in humans [94]. CLR (calcitonin receptor-like receptor) by itself binds no known endogenous ligand, but in the presence of RAMPs it gives receptors for CGRP, adrenomedullin and adrenomedullin 2/intermedin. There are several approved drugs that target this receptor family, such as pramlintide, erenumab, and the "gepant" class of CGRP receptor antagonists.

Adrenomedullin-RAMP2 and -RAMP3 systems regulate cardiac homeostasis during cardiovascular stress

Adrenomedullin (AM) is a peptide hormone with multiple physiological functions, which are regulated by its receptor activity-modifying proteins, RAMP2 and RAMP3. We previously reported that AM or RAMP2 knockout (AM-/-, RAMP2-/-) is embryonically lethal in mice, whereas RAMP3-/- mice are apparently normal. AM, RAMP2 and RAMP3 are all highly expressed in the heart; however, their functions there are not fully understood. Here, we analyzed the pathophysiological functions of the AM-RAMP2 and AM-RAMP3 systems in hearts subjected to cardiovascular stress.Cardiomyocyte-specific RAMP2-/- (C-RAMP2-/-) and RAMP3-/- showed no apparent heart failure at base line. After one week of transverse aortic constriction (TAC), however, C-RAMP2-/- exhibited significant cardiac hypertrophy, decreased ejection fraction and increased fibrosis as compared to wild-type mice. Both dP/dtmax and dP/dtmin were significantly reduced in C-RAMP2-/-, indicating reduced ventricular contractility and relaxation. Exposing CRAMP2/- cardiomyocytes to isoproterenol enhanced their hypertrophy and oxidative stress as compared to wild-type cells. CRAMP2/- cardiomyocytes also contained fewer viable mitochondria and showed reduced mitochondrial membrane potential and respiratory capacity. RAMP3-/- also showed reduced systolic function and enhanced fibrosis after TAC, but those only became apparent after 4 weeks. A reduction in cardiac lymphatic vessels was the characteristic feature in RAMP3-/-.These observations indicate the AM-RAMP2 system is necessary for early adaptation to cardiovascular stress through regulation of cardiac mitochondria. AM-RAMP3 is necessary for later adaptation through regulation of lymphatic vessels. The AM-RAMP2 and AM-RAMP3 systems thus play separate critical roles in the maintenance of cardiovascular homeostasis against cardiovascular stress.

Oral Amylin Treatment Reduces the Pathological Cascade of Alzheimer’s Disease in a Mouse Model

Intraperitoneal injection of amylin or its analog reduces Alzheimer’s disease (AD) pathology in the brains. However, self-injecting amylin analogs is difficult for patients due to cognitive deficits. This work aims to study the effects of amylin on the brain could be achieved by oral delivery as some study reported that amylin receptor may be present in the gastrointestinal tract. A 6-week course of oral amylin treatment reduced components of AD pathology, including the levels of amyloid-β, phosphorylated tau, and ionized calcium binding adaptor molecule 1. The treatment reduced active forms of cyclin-dependent kinase 5. Oral amylin treatment led to improvements in social deficit in AD mouse. Using immunofluorescence, we observed the amylin receptor complexed with the calcitonin receptor and receptor activity-modifying proteins in the enteric neurons. The study suggests the potential of the oral delivery of amylin analogs for the treatment of AD and other neurodegenerative diseases through enteric neurons.

Effects of sub-chronic amylin receptor activation on alcohol-induced locomotor stimulation and monoamine levels in mice

Rationale Amylin receptors consist of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs). The identification of amylin receptors in areas processing reward, namely laterodorsal tegmental area (LDTg), ventral tegmental area (VTA), and nucleus accumbens (NAc), has attributed them a role as reward regulators. Indeed, acute activation of amylin receptors by the amylin receptor agonist salmon calcitonin (sCT) attenuates alcohol-induced behaviours in rodents. Objectives The effects of long-term administration of sCT on alcohol-related behaviours and the molecular mechanisms underlying these processes are not yet elucidated. To fill this knowledge gap, we investigated the effects of sub-chronic sCT treatment on the locomotor stimulatory responses to alcohol in mice and the molecular pathways involved. Methods We assessed the behavioural effects of sub-chronic sCT treatment by means of locomotor activity experiments in mice. We used western blot to identify changes of the CTR levels and ex vivo biochemical analysis to detect changes in monoamines and their metabolites. Results After discontinuation for 5 days of sCT treatment, alcohol did not induce locomotor stimulation in mice pre-treated with sCT when compared with vehicle, without altering secondary behavioural parameters of the locomotor activity experiment or the protein levels of the CTR in reward-related areas in the same set of animals. Moreover, repeated sCT treatment altered monoaminergic neurotransmission in various brain areas, including increased serotonin and decreased dopamine turnover in the VTA. Lastly, we identified a differential effect of repeated sCT and acute alcohol administration on alcohol-induced locomotion in mice, where sCT initially attenuated and later increased this alcohol response. It was further found that this treatment combination did not affect secondary behavioural parameters measured in this locomotor activity experiments. Conclusions These data suggest that sub-chronic sCT treatment differentially alters the ability of alcohol to cause locomotor stimulation, possibly through molecular mechanisms involving various neurotransmitter systems and not the CTR levels per se.

Picomolar affinity antagonist and sustained signaling agonist peptide ligands for the adrenomedullin and calcitonin gene-related peptide receptors

The calcitonin receptor-like G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) actions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) determine CLR ligand selectivity through an unresolved mechanism. CLR-RAMP complexes are drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. We used combinatorial peptide library and rational design approaches to probe selectivity determinants and develop short AM and CGRP variants with ∼1000-fold increased receptor extracellular domain affinities. Binding and structural studies explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in selectivity. In longer scaffolds that also bind the CLR transmembrane domain the variants generated picomolar affinity antagonists, one with an estimated 12.5 hr CGRP receptor residence time, and sustained signaling agonists ss-AM and ss-CGRP. This work clarifies the RAMP-modulated ligand selectivity mechanism and provides AM and CGRP variants with promise as long-acting therapeutics.

Calcitonin receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

This receptor family comprises a group of receptors for the calcitonin/CGRP family of peptides. The calcitonin (CT), amylin (AMY), calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CGRP, AM, AMY, and CT receptors [122, 67]) are generated by the genes CALCR (which codes for the CT receptor) and CALCRL (which codes for the calcitonin receptor-like receptor, CLR, previously known as CRLR). Their function and pharmacology are altered in the presence of RAMPs (receptor activity-modifying proteins), which are single TM domain proteins of ca. 130 amino acids, identified as a family of three members; RAMP1, RAMP2 and RAMP3. There are splice variants of the CT receptor; these in turn produce variants of the AMY receptor [122], some of which can be potently activated by CGRP. The endogenous agonists are the peptides calcitonin, α-CGRP (formerly known as CGRP-I), β-CGRP (formerly known as CGRP-II), amylin (occasionally called islet-amyloid polypeptide, diabetes-associated polypeptide), adrenomedullin and adrenomedullin 2/intermedin. There are species differences in peptide sequences, particularly for the CTs. CTR-stimulating peptide (CRSP) is another member of the family with selectivity for the CT receptor but it is not expressed in humans [87]. olcegepant (also known as BIBN4096BS, pKi~10.5) and telcagepant (also known as MK0974, pKi~9) are the most selective antagonists available, showing selectivity for CGRP receptors, with a particular preference for those of primate origin. CLR (calcitonin receptor-like receptor) by itself binds no known endogenous ligand, but in the presence of RAMPs it gives receptors for CGRP, adrenomedullin and adrenomedullin 2/intermedin.

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Although receptor activity-modifying proteins (RAMPs) have been shown to modulate the functions of several different G protein-coupled receptors (GPCRs), potential direct interactions among the three known RAMPs and hundreds of GPCRs has never been investigated. We engineered three epitope-tagged RAMPs and 23 epitope-tagged GPCRs, focusing on the secretin-like family of GPCRs, and developed a suspension bead array (SBA) immunoassay designed to detect RAMP-GPCR complexes. We then used 64 antibodies raised against native RAMPs and GPCRs, along with four antibodies targeting the epitope tags, to multiplex the SBA assay to detect and measure all possible combinations of interaction among the 23 GPCRs and three RAMPs. The results of the SBA assay provide a complete interactome of secretin-like GPCRs with RAMPs. We demonstrate direct interaction of previously reported secretin-like GPCRs whose functions are modulated by RAMPs. We also discovered novel sets of GPCR-RAMP interacting pairs, and found additional secretin-like GPCRs, chemokine receptors and orphan receptors that interact with RAMPs. Using in situ roximity ligation assay, we verified a subset of these novel GPCR-RAMP interactions in cell membranes. In total, we found GPCR-RAMP interactions for the majority of the 23 GPCRs tested. Each GPCR interacted with either all three RAMPs or with RAMP2 and RAMP3, with the exception of one GPCR that interacted with just RAMP3. In summary, we describe an SBA strategy that will be useful to search for GPCR-RAMP interactions in cell lines and tissues, and conclude that GPCR-RAMP interactions are more common than previously appreciated.