Methods to measure calcitonin receptor activity, up-regulated in cell stress, apoptosis and autophagy [version 1; peer review: 2 approved]
Ontology highlight
ABSTRACT:
SUBMITTER: Wookey P
PROVIDER: S-EPMC8543166 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer.
| S-EPMC5023108 | biostudies-literature
Project description:BACKGROUND AND PURPOSE: Receptor 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 APPROACH: Guided 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 RESULTS: An 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 IMPLICATIONS: RAMP-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.
| S-EPMC3969088 | biostudies-literature
Project description:Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.
| S-EPMC4861438 | biostudies-literature
Project description:Type 1 diabetes is an autoimmune disorder characterized by chronic inflammation and pancreatic beta-cell loss. Here, we demonstrate that the proinflammatory cytokine interleukin-1beta, combined with interferon-gamma, induces the expression of the Bcl-2 homology 3 (BH3)-only activator PUMA (p53 up-regulated modulator of apoptosis) in beta-cells. Transcriptional activation of PUMA is regulated by nuclear factor-kappaB and endoplasmic reticulum stress but is independent of p53. PUMA activation leads to mitochondrial Bax translocation, cytochrome c release, and caspase-3 cleavage resulting in beta-cell demise. The antiapoptotic Bcl-XL protein is localized mainly at the mitochondria of the beta-cells and antagonizes PUMA action, but Bcl-XL is inactivated by the BH3-only sensitizer DP5/Hrk in cytokine-exposed beta-cells. Moreover, a pharmacological mimic of the BH3-only sensitizer Bad, which inhibits Bcl-XL and Bcl-2, induces PUMA-dependent beta-cell death and potentiates cytokine-induced apoptosis. Our data support a hierarchical activation of BH3-only proteins controlling the intrinsic pathway of beta-cell apoptosis in the context of inflammation and type 1 diabetes.
| S-EPMC2888402 | biostudies-literature
Project description:Spectral imaging systems are capable of detecting and quantifying subtle differences in light quality. In this study we coupled spectral imaging with fluorescence and white light microscopy to develop new methods for quantifying autophagy and apoptosis. For autophagy, we employed multispectral imaging to examine spectral changes in the fluorescence of LC3-GFP, a chimeric protein commonly used to track autophagosome formation. We found that punctate autophagosome-associated LC3-GFP exhibited a spectral profile that was distinctly different from diffuse cytosolic LC3-GFP. We then exploited this shift in spectral quality to quantify the amount of autophagosome-associated signal in single cells. Hydroxychloroquine (CQ), an anti-malarial agent that increases autophagosomal number, significantly increased the punctate LC3-GFP spectral signature, providing proof-of-principle for this approach. For studying apoptosis, we employed the Prism and Reflector Imaging Spectroscopy System (PARISS) hyperspectral imaging system to identify a spectral signature for active caspase-8 immunostaining in ex vivo tumor samples. This system was then used to rapidly quantify apoptosis induced by lexatumumab, an agonistic TRAIL-R2/DR5 antibody, in histological sections from a preclinical mouse model. We further found that the PARISS could accurately distinguish apoptotic tumor regions in hematoxylin and eosin-stained sections, which allowed us to quantify death receptor-mediated apoptosis in the absence of an apoptotic marker. These spectral imaging systems provide unbiased, quantitative and fast means for studying autophagy and apoptosis and complement the existing methods in their respective fields.
| S-EPMC3230317 | biostudies-literature
Project description:The aim of this study was to evaluate the clinical significance of N-myc downstream-regulated gene 1 (NDRG1) in colorectal cancer (CRC) patients and to explore the mechanisms governing the role of NDRG1 in apoptosis of CRC cells. In the current study, we found that NDRG1 was a prognostic marker of CRC patients. Moreover, NDRG1 expression negatively correlated to tumor size and clinical TNM stage, suggesting that NDRG1 might act as a tumor suppressor by inhibiting proliferation or inducing apoptosis in CRC. Consistently, substantial apoptosis was observed in vitro and in vivo in the presence of NDRG1. From a mechanistic standpoint, we discovered that NDRG1 was able to prevent death receptor 4 from degradation induced by MARCH-8, a member of the membrane-associated RING-CH (MARCH) ubiquitin ligase family. As a consequence, CRC cells expressing NDRG1 were more sensitive to reagents targeting death receptors such as tumor necrosis factor-related apoptosis-inducing ligands (TRAIL). Additionally, the pro-apoptotic effect of NDRG1 was also validated in mouse xenograft model. In conclusion, our results provided further insights of the pivotal role of NDRG1 in apoptosis initiated by death receptors and demonstrated a novel marker to predict the sensitivity of CRC to TRAIL treatment in future clinical study.
| S-EPMC5669913 | biostudies-literature
Project description:Background and purposeAlternative splicing expands proteome diversity to GPCRs. Distinct receptor variants have been identified for a secretin family GPCR, the calcitonin receptor (CTR). The possible functional contributions of these receptor variants are further altered by their potential interactions with receptor activity-modifying proteins (RAMPs). One variant of the human CTR lacks the first 47 residues at its N terminus [?(1-47)hCT((a)) ]. However, very little is known about the pharmacology of this variant or its ability to interact with RAMPs to form amylin receptors.Experimental approach?(1-47)hCT((a)) was characterized both with and without RAMPs in Cos7 and/or HEK293S cells. The receptor expression (ELISA assays) and function (cAMP and pERK1/2 assays) for up to six agonists and two antagonists were determined.Key resultsDespite lacking 47 residues at the N terminus, ?(1-47)hCT((a)) was still able to express at the cell surface, but displayed a generalized reduction in peptide potency. ?(1-47)hCT((a)) retained its ability to interact with RAMP1 and formed a functional amylin receptor; this also appeared to be the case with RAMP3. On the other hand, its interaction with RAMP2 and resultant amylin receptor was reduced to a greater extent.Conclusions and implications?(1-47)hCT((a)) acts as a functional receptor at the cell surface. It exhibits altered receptor function, depending on whether it associates with a RAMP and which RAMP it interacts with. Therefore, the presence of this variant in tissues will potentially contribute to altered peptide binding and signalling, depending on the RAMP distribution in tissues.
| S-EPMC3579285 | biostudies-literature
Project description:Perfluorooctane sulfonate (PFOS) is among the most abundant organic pollutants and is widely distributed in the environment, wildlife, and humans. Its toxic effects and biological hazards are associated with its long elimination half-life in humans. However, how it affects renal tubular cells (RTCs) remains unclear. In this study, PFOS was observed to mediate the increase in reactive oxygen species (ROS) generation, followed by the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, which induced autophagy in RTCs. Although PFOS treatment induced autophagy after 6 h, prolonged treatment (24 h) reduced the autophagic flux by increasing lysosomal membrane permeability (LMP), leading to increased p62 protein accumulation and subsequent apoptosis. The increase in LMP was visualized through increased green fluorescence with acridine orange staining, and this was attenuated by 3-methyladenine, an autophagy inhibitor. N-acetyl cysteine and an inhibitor of the mitogen-activated protein kinase kinases (U0126) attenuated autophagy and apoptosis. Taken together, these results indicate that ROS activation and ROS-mediated phosphorylated ERK1/2 activation are essential to activate autophagy, resulting in the apoptosis of PFOS-treated RTCs. Our findings provide insight into the mechanism of PFOS-mediated renal toxicity.
| S-EPMC7817024 | biostudies-literature
Project description: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.
| S-EPMC3415637 | biostudies-literature
Project description:Salmon calcitonin (sCT) and human calcitonin (hCT) are pharmacologically distinct. However, the reason for the differences is unclear. Here we analyze the differences between sCT and hCT on the human calcitonin receptor (CT(a)R) with respect to activation of cAMP signaling, β-arrestin recruitment, ligand binding kinetics and internalization. The study was conducted using mammalian cell lines heterologously expressing the human CT(a) receptor. CT(a)R downstream signaling was investigated with dose response profiles for cAMP production and β-arrestin recruitment for sCT and hCT during short term (<2 hours) and prolonged (up to 72 hours) stimulation. CT(a)R kinetics and internalization was investigated with radio-labeled sCT and hCT ligands on cultured cells and isolated membrane preparations from the same cell line. We found that sCT and hCT are equipotent during short-term stimulations with differences manifesting themselves only during long-term stimulation with sCT inducing a prolonged activation up to 72 hours, while hCT loses activity markedly earlier. The prolonged sCT stimulation of both cAMP accumulation and β-arrestin recruitment was attenuated, but not abrogated by acid wash, suggesting a role for sCT activated internalized receptors. We have demonstrated a novel phenomenon, namely that two distinct CT(a)R downstream signaling activation patterns are activated by two related ligands, thereby highlighting qualitatively different signaling responses in vitro that could have implications for sCT use in vivo.
| S-EPMC3958426 | biostudies-literature