The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation ...across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.
•The identification of several amino acid constraints within the second extracellular loop (ECL2) of the GPCR component of the CGRP receptor.•This study provides a platform to begin to formulate some rules for Family B GPCR structure that regulate function.•The study demonstrates a surprising gain of function substitution. Recovering W283H to wild-type (WT) despite the conserved tryptophan and deleterious W283A substitution.•The central and the distal regions of ECL2 have distinct functional roles.•We have shown a linked network of amino acids, likely to represent a shared structural framework at a ligand-receptor interface that is present across the family B GPCRs.
Activation of Orphan Receptors by the Hormone Relaxin Hsu, Sheau Yu; Nakabayashi, Koji; Nishi, Shinya ...
Science (American Association for the Advancement of Science),
01/2002, Volume:
295, Issue:
5555
Journal Article
Peer reviewed
Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor ...has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3′,5′-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.
Key metabolic hormones, such as insulin, leptin, and adiponectin, have been studied extensively in obesity, however the pathophysiologic relevance of the calcitonin family of peptides remains ...unclear. This family includes calcitonin (CT), its precursor procalcitonin (PCT), and alpha calcitonin-gene related peptide (αCGRP), which are all encoded by the gene Calca. Here, we studied the role of Calca-derived peptides in diet-induced obesity (DIO) by challenging Calcr-/- (encoding the calcitonin receptor, CTR), Calca-/-, and αCGRP-/- mice and their respective littermates with high-fat diet (HFD) feeding for 16 weeks. HFD-induced pathologies were assessed by glucose tolerance, plasma cytokine and lipid markers, expression studies and histology. We found that DIO in mice lacking the CTR resulted in impaired glucose tolerance, features of enhanced nonalcoholic steatohepatitis (NASH) and adipose tissue inflammation compared to wildtype littermates. Furthermore, CTR-deficient mice were characterized by dyslipidemia and elevated HDL levels. In contrast, mice lacking Calca were protected from DIO, NASH and adipose tissue inflammation, and displayed improved glucose tolerance. Mice exclusively lacking αCGRP displayed a significantly less improved DIO phenotype compared to Calca-deficient mice. In summary, we demonstrate that the CT/CTR axis is involved in regulating plasma cholesterol levels while Calca, presumably through PCT, seems to have a detrimental effect in the context of metabolic disease. Our study provides the first comparative analyses of the roles of Calca-derived peptides and the CTR in metabolic disease.
Peptide receptor radionuclide therapy (PRRT) is an efficient treatment for gastroenteropancreatic neuroendocrine tumors (GEP NETs), with outstanding overall response rates and survival. However, ...little is known about the particular efficacy regarding bone metastasis (BM).
We retrospectively analyzed a consecutive subgroup of 42 patients with BM of GEP NETs treated with PRRT ((177)Lu-octreotate, 4 intended cycles at 3 monthly intervals 10-14 wk; mean activity per cycle, 8.1 GBq). Availability of restaging and outcome data was required for patient inclusion. Baseline characteristics, including age, tumor origin, performance score, Ki-67 index, tumor load, tumor uptake, plasma chromogranin A, and neuron-specific enolase, were analyzed regarding impact on tumor regression (modified M.D. Anderson criteria) and time to progression. Survival analyses were performed using Kaplan-Meier curves, log-rank test at a significance level of P less than 0.05, and Cox proportional hazards model for uni- and multivariate analyses.
Median follow-up was 32 mo. The observed response of BMs consisted of complete remission in 2 (4.8%), partial remission in 14 (33.3%), minor response in 5 (11.9%), stable disease in 16 (38.1%), and progressive disease in 5 (11.9%) patients. Median progression-free survival and overall survival (OS) were 35 mo (26-44, 95% confidence interval) and 51 mo (37-65, 95% confidence interval), respectively. Patients with responding BMs (complete remission, partial remission, or minor response) exhibited a trend toward better OS (median OS not reached after 53 mo) when compared to nonresponding patients (39 mo, P = 0.076). Only Ki-67 index (>10%) and chromogranin A level (>600 ng/mL) contributed to regression analysis.
BM of GEP NETs is effectively controlled by PRRT, with long progression-free survival and OS. Poor patient condition and multifocality of BMs do not clearly affect treatment efficacy, possibly encouraging the use of PRRT in advanced bone metastatic disease. Larger studies are needed to assess predictors of treatment outcome in these patients.
•A pepducin with a peptide sequence derived from FPR1 inhibits neutrophil function.•The FPR1 pepducin hijacks the closely related FPR2.•The FPR1 pepducin competes with a conventional FPR2 agonist for ...binding.
Pepducins constitute a unique class of G-protein coupled receptor (GPCR) modulating lipopeptides. Pepducins with inhibitory effects on neutrophils could potentially be developed into anti-inflammatory pharmaceuticals. A pepducin with a peptide sequence identical to the third intracellular loop of FPR1 was found to inhibit neutrophil functions including granule mobilization and superoxide production. This FPR1-derived pepducin selectively inhibited signaling and cellular responses through FPR2, but not FPR1 as expected. Binding to the neutrophil surface of a conventional FPR2 agonist is also inhibited. The fatty acid is essential for inhibition and pepducins with shorter peptides lose in potency. In summary, a pepducin designed to target FPR1 was found to hijack FPR2 and potently inhibit neutrophil functions.
Platelet derived growth factor-BB (PDGF-BB) is an important mitogen and cell survival factor during development. PDGF-BB binds PDGF receptor-β (PDGFRβ) to trigger receptor dimerization and tyrosine ...kinase activation. We present the pharmacological and biophysical characterization of a blocking PDGF-BB monoclonal antibody, MOR8457, and contrast this to PDGFRβ. MOR8457 binds to PDGF-BB with high affinity and selectivity, and prevents PDGF-BB induced cell proliferation competitively and with high potency. The structural characterization of the MOR8457-PDGF-BB complex indicates that MOR8457 binds with a 2:1 stoichiometry, but that binding of a single MOR8457 moiety is sufficient to prevent binding to PDGFRβ. Comparison of the MOR8457-PDGF-BB structure with that of the PDGFRβ-PDGF-BB complex suggested the potential reason for this was a substantial bending and twisting of PDGF-BB in the MOR8457 structure, relative to the structures of PDGF-BB alone, bound to a PDGF-BB aptamer or PDGFRβ, which makes it nonpermissive for PDGFRβ binding. These biochemical and structural data offer insights into the permissive structure of PDGF-BB needed for agonism as well as strategies for developing specific PDGF ligand antagonists.
Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and ...cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of 18O/16O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using 18O/16O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.
Signal recognition particle (SRP) and its receptor (SR) comprise a highly conserved cellular machine that cotranslationally targets proteins to a protein-conducting channel, the bacterial SecYEG or ...eukaryotic Sec61p complex, at the target membrane. Whether SecYEG is a passive recipient of the translating ribosome or actively regulates this targeting machinery remains unclear. Here we show that SecYEG drives conformational changes in the cargo-loaded SRP-SR targeting complex that activate it for GTP hydrolysis and for handover of the translating ribosome. These results provide the first evidence that SecYEG actively drives the efficient delivery and unloading of translating ribosomes at the target membrane.
A web application, DesMol2, which offers two main functionalities, is presented: the construction of molecular libraries and the calculation of topological indices. These functionalities are ...explained through a practical example of research of active molecules to the formylpeptide receptor (FPR), a receptor associated with chronic inflammation in systemic amyloidosis and Alzheimer's disease. Starting from a data(base) of 106 dioxopiperazine pyrrolidin piperazine derivatives and their respective constant values of binding affinity to FPR, multilinear regression and discriminant analyses are performed to calculate several predictive topological-mathematical models. Next, using the DesMol2 application, a molecular library consisting of 6,120 molecules is built and performed for each predictive model. The best potential active candidates are selected and compared with results from other previous works.
Targeted drug delivery is especially important in cancer treatment as many anti‐cancer drugs are non‐specific and highly toxic to both cancer and normal cells. The targeted drug delivery of DOX to ...the MUC1‐expressing breast cancer cell line (MCF7) was obtained using APT as a carrier. Modification of the APT‐DOX complex by PEG increases the survivability of the macrophage control (RAW 264.7) by about six‐fold as compared to free DOX treatment without significantly affecting the cytotoxicity toward the target cell line. Thus, PEG‐APT‐DOX is potentially a new therapeutic agent for targeted drug delivery to MUC1‐expressing cell lines.
DOX intercalates APT without significantly affecting either the cytotoxicity of DOX or the targeting efficacy of APT. The PEGylated APT‐DOX complex presents significant cytotoxicity against MUC1‐overexpressing breast cancer cells (MCF7), while minimizing adverse impact on the macrophages RAW 264.7. Thus, the PEG‐APT‐DOX complex can potentially be used to combat MUC1‐expressing cancers.
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