Gareth O. Richards

Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1tr/tr) have ~50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1tr/tr osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1tr/tr cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.

Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)

Background: The glucagon and glucagon-like peptide-1 (GLP-1) receptors are important targets for treating type 2 diabetes. Results: We describe novel glucagon receptor pharmacology, through interaction with the receptor activity-modifying protein-2 (RAMP2). Conclusion: RAMP2 regulates both ligand binding and G protein selectivity of the glucagon receptor. Significance: The effect of RAMP2 should be considered when designing anti-diabetic treatments. The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.

Research Resource: Haploinsufficiency of Receptor Activity-Modifying Protein-2 (Ramp2) Causes Reduced Fertility, Hyperprolactinemia, Skeletal Abnormalities, and Endocrine Dysfunction in Mice

Receptor activity-modifying protein-2 (RAMP2) is a single-pass transmembrane protein that can regulate the trafficking, ligand binding, and signaling of several G protein-coupled receptors (GPCR). The most well-characterized role of RAMP2 is in the regulation of adrenomedullin (AM) binding to calcitonin receptor-like receptor (CLR), and our previous studies using knockout mouse models support this canonical signaling paradigm. For example, Ramp2(-/-) mice die at midgestation with a precise phenocopy of the AM(-/-) and Calcrl(-/-) mice. In contrast, Ramp2(+/-) mice are viable and exhibit an expanded variety of phenotypes that are distinct from those of Calcrl(+/-) mice. Using Ramp2(+/-) female mice, we demonstrate that a modest decrease in Ramp2 expression causes severe reproductive defects characterized by fetal growth restriction, fetal demise, and postnatal lethality that is independent of the genotype and gender of the offspring. Ramp2(+/-) female mice also exhibit hyperprolactinemia during pregnancy and in basal conditions. Consistent with hyperprolactinemia, Ramp2(+/-) female mice have enlarged pituitary glands, accelerated mammary gland development, and skeletal abnormalities including delayed bone development and decreased bone mineral density. Because RAMP2 has been shown to associate with numerous GPCR, it is likely that signaling of one or more of these GPCR is compromised in Ramp2(+/-) mice, yet the precise identification of these receptors remains to be elucidated. Taken together, this work reveals an essential role for RAMP2 in endocrine physiology and provides the first in vivo evidence for a physiological role of RAMP2 beyond that of AM/CLR signaling.

Role of Receptor Activity Modifying Protein 1 in Function of the Calcium Sensing Receptor in the Human TT Thyroid Carcinoma Cell Line

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca2+ and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred.

The Role of the Calcitonin Peptide Family in Prostate Cancer and Bone Metastasis

Purpose of ReviewThis study is to highlight recent discoveries associated with the role of calcitonin peptide family and their receptors in prostate cancer progression and bone metastasis.Recent FindingsStudies have linked adrenomedullin (AM), calcitonin (CT) and calcitonin gene-related peptide (CGRP) to the spread of prostate tumours to the bone. AM can induce a metastatic phenotype in prostate cancer cells through its action on TRPV2 calcium channels and is also capable of influencing localised levels of RANKL in the bone to favour tumourigenesis. CT utilises A-kinase anchoring proteins to indirectly act on PKA and promote metastasis in prostate cancer. The receptor for CT contains a PDZ-binding domain, the deletion of which stops metastasis to the bone in orthotopic prostate models.SummaryRecent findings show strong evidence for the role of calcitonin peptides and receptors in prostate cancer and bone metastasis. Further research could provide potential prognostic markers and therapeutic targets for prostate cancer patients.

Role of receptor activity modifying protein 3 in the response of bone to mechanical loading

Introduction Receptor Activity Modifying Protein 3 (RAMP3) is a single pass trans-membrane domain protein which interacts with the calcitonin receptor (CTR) and calcitonin-like receptor (CLR) in order to aid trafficking to the cell surface and to change ligand selectivity. The adaptive responses of the skeleton to loading changes the architecture and physical properties, in order to optimise strength for function. However, bone is subjected to many local and circulating osteotropic factors, many acting on G-protein coupled receptors, a family to which the CTR and CLR belong. Previous work in our laboratory has shown that RAMP3 knock-out mice have a high bone bass phenotype and a leaner body mass through to old age.