Date of Award

2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

College

College of Graduate Studies

First Advisor

Lauren E. Ball

Second Advisor

Louis M. Luttrell

Third Advisor

Joe B. Blumer

Fourth Advisor

Keith L. Kirkwood

Fifth Advisor

David Kurtz

Sixth Advisor

Gerard T. Hardiman

Abstract

The past 20 years have seen G-protein coupled receptor (GPCR) theory advance significantly. Receptors are now thought of as adopting multiple conformations in a state of dynamic equilibrium. The study of GPCR biased agonism has emerged from this changing concept of receptors and introduced the field to “pluridimensional efficacy.” It is thought that a single readout of efficacy is no longer sufficient and multiple parameters of efficacy must be measured in drug screens to improve the ability to predict in vivo effects. While several GPCRs have multiple cognate ligands that elicit functionally-selective responses, the present study focused on biased signaling of the parathyroid hormone receptor (PTH1R). Parathyroid hormone (PTH) maintains serum calcium and is a key regulator of bone remodeling. Human PTH1-34 (Forteo) is the only FDA approved drug used for treatment of osteoporosis that acts via its anabolic actions on osteoblasts. However, the therapeutic utilization of PTH1-34 is limited by its catabolic effects, mediated in part by protein kinase A, which after two years culminate in net bone resporption through the activation of osteoclasts by RANKL. The experimental, biased ligand of the PTH1R, bovine parathyroid hormone residues 7-34 with D-Trp12 and Tyr34 (bPTH(7-34)), does not exhibit the catabolic effects of PTH1-34. In vivo administration of the conventional ligand, PTH 1-34, and the biased ligand, bPTH(7-34), for eight weeks increased bone mineral density (BMD) in mice. The anabolic effect of bPTH(7-34) in vivo was lost in β-arrestin deficient mice, revealing a dependence on β-arrestin mediated signaling. Further analysis of osteoblast and osteoclast number, transcript expression, and the generation of second messengers revealed the anabolic effect of each ligand was achieved by different mechanisms. To elucidate the unique, proximal signaling events activated by acute stimulation of the PTH1R with the biased agonist, this study focused on characterization and comparison of the phosphorylation-mediated signaling profiles of osteoblasts stimulated with each osteogenic agonist. Relative changes in phosphorylation were measured using a SILAC-based phosphoproteomic screen following acute stimulation of MC3T3- E1 preosteoblast cells with hPTH(1-34) or bPTH(7-34) for 5 minutes. The experiments were performed in proliferating preosteoblasts (Day 0) and differentiating osteoblasts (Day 10). Over ten thousand sites of phosphorylation were observed. Regulated phosphosites and phosphoproteins were examined for putative kinase activity, targeted signaling pathways, and biological processes. Differences were observed in the kinases stimulated by each agonist. For example, bPTH(7-34) treatment activated MAPK1 and increased phosphorylation of downstream substrates, while phosphorylation of predicted MAPK1 substrates were decreased with hPTH(1-34) activation. While both drugs regulated phosphorylation of proteins in signaling pathways involving GPCR signaling (PLC, MTOR, Rho GTPases); Ingenuity Pathway Analysis (IPA) also revealed discrete signaling networks engaged by each drug. PTH (1-34) treatment yielded regulated proteins involved in cytoskeletal dynamics and the Wnt/β- catenin pathway, whereas bPTH(7-34) treatment modulated pathways related to survival (ATM, CDKs, and p70S6K) and transcription (Jak/Stat, and PPARα). Cell-based assays confirmed hPTH(1-34) and bPTH(7-34) both confer resistance to etoposide-induced apoptosis and bPTH(7-34) increases proliferation in MC3T3-E1 cells. At the biological process level, both ligands modulated proteins involved in cell survival, migration, growth, and bone metabolism. Comparison of regulated phosphoproteins at two time points during osteogenic differentiation unexpectedly revealed that the bPTH(7-34) gave a more robust effect in proliferating preosteoblasts, whereas hPTH(1-34) stimulated more sites of phosphorylation in differentiating osteoblasts. This observation indicates the differential effects of each agonist may result from changes in signaling mediators that are expressed at these two time points. While the PTH receptor was present at both time points, β-arrestin was more highly expressed in proliferating preosteoblasts.

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