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Title: beta-Arrestin Pathway-Selective G Protein-Coupled Receptor Agonists Engender Unique Biological Efficacy In Vivo      
keywords:
Transcriptome or Gene expression
ID:
PRJNA150669
description:
Biased GPCR agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, D-Trp12,Tyr34-bPTH(7-34) (PTH-{beta}arr), a biased agonist for the type 1 parathyroid hormone receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both PTH-{beta}arr and the conventional agonist PTH(1-34) stimulate anabolic bone formation. To understand how two PTH1R ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 weeks with vehicle, PTH-{beta}arr or PTH(1-34). Treatment of wild type mice with PTH-{beta}arr primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival and migration. These responses were absent in beta-arrestin2 null mice, identifying them as downstream targets of beta-arrestin2-mediated signaling. In contrast, PTH(1-34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. PTH(1-34) actions were less dependent on beta-arrestin2, as might be expected of a ligand capable of G protein activation. These results illustrate the uniqueness of biased agonism in vivo and demonstrate that functional selectivity can be exploited to change the quality of GPCR efficacy. Overall design: The derivation of beta-arrestin2 null mice was previously described. Mice were backcrossed for greater than nine generations onto a C57BL/6J background. Either Human PTH(1–34) (40 µg/kg•d), bovine (D-Trp12, Tyr34)-PTH(7-34) (40 µg/kg•d) or PBS vehicle was administered to nine week-old male mice via intraperitoneal injection, daily for 8 weeks. Total cellular RNA was isolated from the calvaria of the treated mice and RNA was isolated according to the TRIzol manufacturer’s protocol. The total RNA was analyzed for concentration (ng/uL) and purity (ratios of 260/280 nm and 260/230 nm) using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). RNA integrity was analyzed using the Experion RNA HighSens Analysis Kit (Bio-Rad Laboratories, Inc., Hercules CA). Hybridization targets were prepared from this total RNA using the Affymetrix one cycle kit (Cat#900431) which creates biotinylated cRNA. The labeled cRNA samples were hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) in the Duke Microarray Facility of the Duke Institute for Genome Sciences & Policy. Three replicates were performed for each condition. Raw hybridization intensity data were log transformed and normalized to yield Z-scores, which in turn were used to calculate a Z-ratio value for each gene with respect to the control geneset. The Z-ratio was calculated as the difference between the observed gene Z-scores for the experimental and the control comparisons divided by the standard deviation associated with the distribution of these differences.
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA150669
authentication:
none
authorization:
none
ID:
pmid:23315939
name:
Mus musculus
ncbiID:
ncbitax:10090
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject