Elsevier

Methods in Enzymology

Volume 522, 2013, Pages 229-262
Methods in Enzymology

Chapter Thirteen - Biasing the Parathyroid Hormone Receptor: Relating In Vitro Ligand Efficacy to In Vivo Biological Activity

https://doi.org/10.1016/B978-0-12-407865-9.00013-3Get rights and content

Abstract

Recent advances in our understanding of the pluridimensional nature of GPCR signaling have provided new insights into how orthosteric ligands regulate receptors, and how the phenomenon of functional selectivity or ligand “bias” might be exploited in pharmaceutical design. In contrast to the predictions of simple two-state models of GPCR function, where ligands affect all aspects of GPCR signaling proportionally, current models assume that receptors exist in multiple “active” conformations that differ in their ability to couple to different downstream effectors, and that structurally distinct ligands can bias signaling by preferentially stabilizing different active states. The type 1 parathyroid hormone receptor (PTH1R) offers unique insight into both the opportunities and challenges of exploiting ligand bias in pharmaceutical design, not only because numerous “biased” PTH analogs have been described but also because many of them have been characterized for biological activity in vivo. The PTH1R has pleiotropic signaling capacity, coupling to Gs, Gq/11, and Gi/o family heterotrimeric G proteins, and binding arrestins, which mediate receptor desensitization and arrestin-dependent signaling. Here, we compare the activity of six different PTH1R ligands in a common HEK293 cell background using three readouts of receptor activation, cAMP production, intracellular calcium influx, and ERK1/2 activation, demonstrating the range of signal bias that can be experimentally observed in a “typical” screening program. When the in vitro activity profiles of these ligands are compared to their reported effects on bone mass in murine models, it is apparent that ligands activating cAMP production produce an anabolic response that does not correlate with the ability to also elicit calcium signaling. Paradoxically, one ligand that exhibits inverse agonism for cAMP production and arrestin-dependent ERK1/2 activation in vitro, (D-Trp12, Tyr34)-bPTH(7–34), reportedly produces an anabolic bone response in vivo despite an activity profile that is dramatically different from that of other active ligands. This underscores a major challenge facing efforts to rationally design “biased” GPCR ligands for therapeutic application. While it is clearly plausible to identify functionally selective ligands, the ability to predict how bias will affect drug response in vivo, is often lacking, especially in complex disorders.

Introduction

Early efforts to model the action of drugs or hormones assumed that individual receptors behave as binary switches, existing in equilibrium between an “off” state, which is silent in the assay, and an “on” state, which is capable of generating a measurable response. In such models, receptor conformation is the minimal determinant of system response and ligands act solely by changing the fraction of the receptor population in the on state (Karlin, 1967, Thron, 1973). The efficacy of a ligand thus becomes a reflection of its ability to stabilize the on state and can be approximated by two parameters: the maximal observed response (Emax) and potency (EC50), the ligand concentration that produces a half-maximal response. In this context, full agonists are ligands that preferentially bind and stabilize the on state, producing the maximum system response at saturating ligand concentration; partial agonists are ligands with less conformational selectivity, translating into a submaximal system response at saturating concentration and potential attenuation of full agonist activity; true neutral antagonists are ligands with equal affinity for both the off and on conformations, producing no physiological response but able to block the response to agonists; and inverse agonists are ligands that preferentially bind the off state, which causes them to appear as antagonists in systems with low basal activity but with the added property of reducing receptor-mediated constitutive activity in systems with high basal tone.

Although readily determined experimentally, measurements of EC50 and Emax are limited in that they are influenced by system factors external to the ligand–receptor unit (Ehlert, 2000, Figueroa et al., 2009). In the case of G protein-coupled receptors (GPCRs), differences in receptor reserve and signal amplification can lead to apparent changes in ligand classification when comparisons are made between different assays. New signaling responses commonly emerge as the level of receptor expression increases, permitting less efficiently coupled effectors to reach the detection threshold of the assay (Zhu, Gilbert, Birnbaumer, & Birnbaumer, 1994). Similarly, variation in the expression levels of G proteins, arrestins, and downstream effectors can make ligand activity appear to change between cell types (Nasman, Kukkonen, Ammoun, & Akerman, 2001). Even in the same cell background, ligands may appear as full agonists when classified using signals that are highly amplified, for example, cAMP production, but as partial agonists when assayed for less amplified responses, for example, arrestin binding and signaling (Rajagopal et al., 2010).

Nonetheless, signal strength arguments cannot account for true reversal of potency or efficacy, for example, when the rank order of potency for two ligands acting on the same receptor is opposite in two different assays of cellular response (Berg et al., 1998). In a two-state model, ligand binding can alter the fraction of receptors in the on state, but cannot qualitatively change the nature of that state. Thus, the classification of a ligand as an agonist, antagonist, or inverse agonist must be independent of the assay used to detect receptor activation, and the rank order of potency for a series of ligands cannot vary when two or more assays are employed. Reversal of potency or efficacy implies that different ligands are activating the same receptor in different ways, meaning that they must be generating different active receptor states (Kenakin, 1995). That this phenomenon has now been described for several GPCRs, among them the serotonin 5-HT2c, pituitary adenylate cyclase-activating polypeptide, dopamine D2, neurokinin NK1, CB1 cannabinoid, β2 adrenergic, angiotensin AT1A, and PTH1Rs, suggests that most, if not all, GPCRs can adopt multiple active conformations (Luttrell & Kenakin, 2011).

It is now apparent that GPCR signaling is “pluridimensional” (Galandrin & Bouvier, 2006), meaning that receptors signal by coupling to multiple G protein and non-G protein effectors. If different active conformations couple the receptor to these downstream effectors with different efficiency, then the cellular response will be dictated by the distribution of receptors across the range of achievable active and inactive states. Since there is no a priori reason that the active conformation(s) favored by one ligand should be identical either to the spontaneously formed active state or to those preferred by a structurally distinct ligand, the potential exists for the ligand to bias signaling in favor of some effectors at the expense of others. Thus, it is the ligand–receptor complex, not the receptor alone, that specifies the active state, along with any other small molecule, protein–protein, or lipid–protein interaction that allosterically constrains the conformations available to the receptor (Kenakin & Miller, 2010). In contrast to a two-state model, wherein agonists and antagonists merely control the quantity of receptor activity, the potential of biased agonism lies in its ability to qualitatively change signaling.

Here, we employ the PTH1R to illustrate some of the issues arising from pluridimensional efficacy and the challenge of adequately describing ligand bias. Using a selected panel of PTH analogs and a multiplexed set of cell-based assays for cAMP, intracellular calcium release, and ERK1/2 activation, we demonstrate the assay-dependence of efficacy and the range of signaling responses achievable through biased agonism. These results are then discussed in the context of the known effects of these same ligands in vivo to illustrate the difficulty and complexity of using in vitro profiles of ligand activity to predict biological response.

Section snippets

Determining the Relative Activity of PTH1R Ligands

For any given GPCR ligand, an activity profile can be generated by determining its EC50 and Emax across a panel of assays measuring different indices of receptor activation. These results will be specific for each receptor–ligand combination, but they will also be subject to system factors influencing coupling efficiency, such as receptor density, that can cause ligand classification to appear to vary between assays. Such factors cannot change the relative order of potency or efficacy for a

Discussion

The principal targets of PTH in vivo are kidney and bone, where its actions promote a rise in serum calcium. In the kidney, PTH stimulates reabsorption of filtered calcium by the cortical thick ascending limb of the loop of Henle and distal convoluted tubule. In the proximal tubule, it stimulates expression of the 1α-hydroxylase that converts 25(OH)-vitamin D to its active form 1,25(OH)2-vitamin D, which in turn enhances intestinal calcium absorption. The actions of PTH in bone are complex. PTH

Acknowledgments

The authors thank Dr. Diane Gesty-Palmer (Duke University Medical Center) for helpful advice and criticism and Allie Pinosky for technical assistance. The work was supported by National Institutes of Health Grant R01 DK55524 (L. M. L.), the MUSC fluorescence imaging plate reader (FLIPRtetra) facility (S10 RR027777; L. M. L./T. A. M.), and the Research Service of the Charleston, SC Veterans Affairs Medical Center (L. M. L./T. A. M.). The contents of this chapter do not represent the views of the

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