I Physiological Functions of Biased Signaling at the Chemokine

Physiological Functions of Biased Signaling at the Chemokine Receptor CXCR3

Jeffrey Scott Smith

Department of Biochemistry Duke University

Date:______Approved:

______Sudarshan Rajagopal, Supervisor

______Robert J. Lefkowitz

______Margarethe J. Kuehn

______Terrence G. Oas

______Russell P. Hall III

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University

2018

ABSTRACT

Physiological Functions of Biased Signaling at the Chemokine Receptor CXCR3

Jeffrey Scott Smith

Department of Biochemistry Duke University

Date:______Approved:

______Sudarshan Rajagopal, Supervisor

______Robert J. Lefkowitz

______Margarethe J. Kuehn

______Terrence G. Oas

______Russell P. Hall III

An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University

2018

Abstract

G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and one of the most common drug targets. It is now well-established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, G protein receptor kinases, and beta-arrestins. Certain ligands can preferentially activate certain signaling cascades while inhibiting others, a phenomenon referred to as biased signaling. While biased signaling is observed in many ex-vivo assays, the physiological relevance of biased signaling is not well established. Using the chemokine receptor CXCR3, a receptor that regulates T cell function, and its endogenous chemokines CXCL9, CXCL10, and CXCL11, I established that endogenous biased signaling exists at CXCR3. After identifying small molecule biased CXCR3 agonists using cell-based assays, I utilized human samples and mouse models of T cell movement and inflammation to determine that differential activation of either the G protein or beta-arrestin signaling pathways downstream of CXCR3 produces distinct functional differences. I identified that beta-arrestin regulated-Akt signaling appears critical for full efficacy chemotaxis. I conclude that biased signaling at CXCR3 produces distinct physiological responses.

Dedication

To my mother and father, Elaine G. Chapman and Richard D. Smith, for their boundless support and love.

Contents

Abstract ...... iv

List of Tables ...... xi

List of Figures ...... xii

Acknowledgements ...... xv

1. Introduction ...... 1

1.1 Overview of G protein Coupled Receptors ...... 1

1.2 Overview of beta-arrestins ...... 3

1.2.1 Beta-arrestin signaling roles ...... 3

1.3 A primer on biased signaling ...... 4

1.3.1 The ternary complex: a validated model for assessing GPCR signaling ...... 5

1.3.2 Modifying components of a ternary complex can bias signaling...... 7

1.3.3 Transmission of ligand bias ...... 10

1.4 Phosphorylation ‘bar codes’ and human disease ...... 11

1.5 Structural basis of biased agonism ...... 13

1.6 Emerging strategies for determining GPCR structure ...... 16

1.7 Beta-arrestins ...... 19

1.7.1 Distinct and Overlapping Roles for the beta-arrestins ...... 21

1.7.2 Beta-arrestin post-translational modifications ...... 22

1.7.3 Beta-arrestin-regulated Desensitization ...... 23

1.7.4 Beta-arrestin-regulated Receptor Trafficking ...... 25

1.7.5 Beta-arrestin-regulated receptor signaling ...... 27

1.7.6 Beta-arrestin-biased agonism ...... 29

1.7.7 The signaling barcode: A model for allosteric regulation of beta-arrestins ..... 32

1.7.8 Signal transduction to effectors ...... 33

2. Quantifying Bias ...... 34

2.1 Historical context of quantifying receptor signalling ...... 34

2.1.1 Assays for detecting G protein and beta-arrestin signaling ...... 35

2.1.2 Identifying Biased Ligands Qualitative assessment ...... 40

2.2 Translating in vitro bias into in vivo utility ...... 42

2.3 Biased Signaling at Chemokine Receptors ...... 45

3. CXCR3 splice variants differentially activate beta-arrestins to regulate downstream signaling pathways ...... 46

3.1 CXCR3 receptor splice variants ...... 46

3.2 CXCR3B is beta-arrestin-biased compared to CXCR3A ...... 48

3.3 Both CXCR3A and CXCR3B undergo agonist-dependent phosphorylation ...... 54

3.4 Activation of CXCR3A and B generate distinct patterns of beta-arrestin recruitment and conformation ...... 55

3.5 CXCR3 splice variants differentially internalize ...... 60

3.6 C-terminus truncation and GRK knockdown reduce beta-arrestin-2 recruitment63

3.7 CXCL11 activates ERK 1/2 at both CXCR3A and CXCR3B ...... 64

3.8 CRISPR/Cas9 knockout of beta-arrestins differentially affects CXCR3 splice variants ...... 67

3.9 Differential transcriptional regulation by CXCR3 splice variants ...... 72

vii

3.10 Discussion of CXCR3 splice variants ...... 74

3.11 Summary of differences between CXCR3 splice variants ...... 78

4. CXCR3 in Physiology and Disease ...... 79

4.1 Introduction to physiological processes regulated by CXCR3 ...... 79

4.2 Allergic contact dermatitis: a disease model for CXCR3 signaling ...... 82

4.2.1 Summary of allergic contact dermatitis ...... 83

4.2.2 Hapten activation of the adaptive immune system initiates allergic contact dermatitis ...... 84

4.2.3 T cells are critical regulators of allergic contact dermatitis ...... 86

4.2.4 The chemokine receptor system as a therapeutic target for allergic contact dermatitis ...... 88

4.2.5 CXCR3 signaling in patients with allergic contact dermatitis ...... 90

4.2.6 CXCR3 signaling in murine models of allergic contact dermatitis ...... 91

5. Biased CXCR3 agonists differentially control chemotaxis and inflammation ...... 91

5.1 Identification of CXCR3 Biased Agonists ...... 92

5.2 A CXCR3 beta-arrestin-biased agonist potentiates T cell-mediated inflammation and chemotaxis ...... 96

5.3 Patients positive for contact hypersensitivity coexpress CXCR3 and beta-arrestin in T cells in the skin ...... 107

5.4 beta-arrestins activate Akt and regulate T cell chemotaxis...... 110

6. Discussion ...... 116

6.1 Implications for CXCR3 Drug Design ...... 116

6.2 Beta-arrestin / Akt regulation of T cell chemotaxis ...... 117

viii

7. Materials and methods ...... 120

7.1.1 Cell Culture ...... 120

7.1.2 Transfections ...... 120

7.1.3 Bioluminescence Resonance Energy Transfer (BRET) ...... 121

7.1.4 GloSensor cAMP Assay ...... 122

7.1.5 TGF-alpha shedding assay ...... 123

7.1.6 Small molecules and peptides ...... 124

7.2 Generation of FLAG-tagged CXCR3 and C-terminal Truncation Mutants ...... 124

7.3.1 GRK knockdown ...... 125

7.3.2 DiscoverX Active Internalization Assay ...... 125

7.3.3 SRE/SRF pathway assay ...... 125

7.4.1 Immunoblot ...... 126

7.4.2 Intact Cell Phosphorylation...... 127

7.4.3 Generation of beta-arrestin1/2-double knockout (∆ARRB1/2) HEK293 cells. 129

7.4.4 Flow Cytometry to assess receptor surface expression ...... 129

7.5.1 Study subjects and skin samples...... 130

7.5.2 Human subjects ...... 130

7.5.3 Mice ...... 131

7.6.1 Mouse allergic contact hypersensitivity (CHS) ...... 131

7.6.2 Chemotaxis transwell assay ...... 132

7.6.3 Skin assessment of T cells ...... 133

7.6.4 Immunofluorescence of human skin ...... 134

7.6.5 Targeted phosphoprotein analysis ...... 135

7.7 Statistical Analyses ...... 136

8. Conclusions ...... 136

References ...... 138

Biography ...... 173

List of Tables

Table 1: Selected assays for assessing ligand bias ...... 36

Table 2: Selected biased agonists under different phases of evaluation for therapeutic use...... 44

Table 3: Efficacies and potencies for chemokines at CXCR3A and CXCR3B...... 50

Table 4: Pharmacological properties of CXCR3 biased agonists...... 95

List of Figures

Figure 1: Biased signaling can be encoded through three general mechanisms...... 6

Figure 2: Drug discovery strategies and physiological consequences of biased signaling...... 12

Figure 3: General approach to characterizing biased ligands...... 41

Figure 4: CXCR3A and CXCR3B splice variants...... 47

Figure 5: Differential G protein activation and beta-arrestin recruitment by CXCR3 splice variants...... 49

Figure 6: Bias plot at CXCR3A...... 53

Figure 7: Differential duration of beta-arrestin-receptor association...... 54

Figure 8: CXCR3A and CXCR3B demonstrate equivalent agonist-induced phosphorylation...... 56

Figure 9: CXCR3A and CXCR3B induce distinct beta-arrestin-2 trafficking patterns...... 57

Figure 10: Limiting beta-arrestin-2 biosensor avidity...... 58

Figure 11: CXCR3A, but not CXCR3B, stimulation with CXCL11 causes a change in beta- arrestin-2 conformation...... 59

Figure 12: Receptor internalization...... 61

Figure 13: GRK5/6 knockdown differentially attenuates beta-arrestin2 recruitment. (A) Cartoon of CXCR3 truncation C-terminal mutants...... 64

Figure 14: Divergent ERK activation kinetics between CXCR3 splice variants...... 66

Figure 15: Confirmation of beta-arrestin knockout...... 68

Figure 16: Beta-arrestin knockout differentially alters pERK signaling between CXCR3 splice variants...... 69

xii

Figure 17: Beta-arrestin overexpression differentially regulates pERK signaling between CXCR3 splice variants...... 71

Figure 18: CXCL11 robustly activates serum response element (SRE) and serum response factor (SRF) response element signaling at CXCR3A, but not CXCR3B...... 73

Figure 19: Summary of observed CXCR3 isoform signaling...... 78

Figure 20: VUF10661 and VUF11418 are CXCR3 biased agonists...... 94

Figure 21: Additional signaling analyses of CXCR3 ligands...... 97

Figure 22: Biased signaling is conserved at murine CXCR3...... 99

Figure 23: A beta-arrestin-biased, but not G protein-biased, CXCR3 agonist increases inflammation and chemotaxis of effector/memory T cells...... 101

Figure 24: The inflammatory effects of the CXCR3 beta-arrestin-biased agonist VUF10661 were absent in CXCR3 KO mice...... 102

Figure 26: Biased CXCR3 ligands differentially increased CD4+CD44+ and total T cells to DNFB-treated ears...... 106

Figure 27: Human CXCR3+ T cells display differential responses to CXCR3 biased agonists while CXCR3 and beta-arrestin are co-expressed in T cell clusters within lesions from human allergen patch tested skin...... 108

Figure 28: Human T cell chemotaxis. Human T cell chemotaxis towards CXCR3 biased agonists...... 109

Figure 29: Akt activation is dependent on beta-arrestin-2 and promotes CXCR3- mediated chemotaxis...... 111

Figure 30: Diverging Akt, but not ERK 1/2, phosphorylation following stimulation with CXCR3 biased agonists in a T cell line stably expressing CXCR3...... 113

xiii

Figure 31: Both pertussis toxin and PI3K inhibition eliminated effector T-cell migration to VUF10661...... 115

xiv

Acknowledgements

I would to thank my primary advisor, Dr. Sudarshan Rajagopal, for his guidance, mentorship and for taking me in as his first graduate student, and my co-mentor, Dr.

Robert J. Lefkowitz, for his guidance and incredible support in all aspects of life. Both are physician-scientists of the highest caliber, and each have helped me grow both as a scientist and as a person. I would also like to acknowledge the mentorship of Dr.

Charles Chavkin, with whom I worked with as an undergraduate and who ignited my passion for scientific discovery. A special acknowledgement is due to the Duke Medical

Scientist Training Program leadership, Dr. Chris Kontos, Dr. Dona Chikaraishi, and

Andrea Liu for opening the door to the incredible scientific opportunities available at

Duke. I believe investigating challenging biomedical research questions is best accomplished as a team, and I was fortunate to work closely with a number of very talented younger trainees: Priya Alagesan, Jaimee Gundry, Nicole Knape, Nimit Desai,

Rachel Glenn, Kevin Zheng, and Issac Choi. These individuals have been phenomenal scientific teammates, as well as friends. All have bright futures in the paths they choose.

I would like to acknowledge Dr. Thomas Pack for his scientific guidance, discussion, and a number of productive collaborations. I would also like to thank the entire

Rajagopal and Lefkowitz laboratories. Finally, I would like to thank my thesis committee members for their support and wisdom.

1. Introduction

Portions of this dissertation were originally published in the Journal of Biological

Chemistry: Smith, Jeffrey S., and Sudarshan Rajagopal. "The β-arrestins: multifunctional regulators of G protein-coupled receptors." Journal of Biological Chemistry 291, no. 17

(2016): 8969-8977 © the American Society for Biochemistry and Molecular Biology; in

Molecular Pharmacology: Smith, Jeffrey S., Priya Alagesan, Nimit K. Desai, Thomas F.

Pack, Jiao-Hui Wu, Asuka Inoue, Neil J. Freedman, and Sudarshan Rajagopal. "CXC motif chemokine receptor 3 splice variants differentially activate beta-arrestins to regulate downstream signaling pathways." Molecular pharmacology 92, no. 2 (2017):

Smith, Jeffrey S., Robert J. Lefkowitz, and Sudarshan Rajagopal. "Biased signaling: from simple switches to allosteric microprocessors." Nature Reviews Drug Discovery (2018).

Other portions, where I am also listed as first author, are currently under review at journals where authors performing the original work currently retain permission for use in academic theses or dissertations.

1.1 Overview of G protein Coupled Receptors

G protein-coupled receptors (GPCRs) are the most common receptors encoded in the genome, comprising greater than 1% of the coding human genome with approximately 800 members and expressed within every organ system [1]. All GPCRs

share a common architecture consisting of an extracellular N-terminal sequence, seven transmembrane-spanning (TM) domains (TM1–TM7) that are connected by three extracellular and three intracellular loops, and an intracellular C-terminal domain. As they regulate virtually every aspect of physiology, it is unsurprising that GPCRs are also the target of > 30% of all prescription drug sales [2, 3]. GPCRs are sensors of a wide array of extracellular stimuli, including proteins, hormones, small molecules, neurotransmitters, ions, and light. GPCR signaling is primarily controlled by interactions with three protein families: G proteins, G protein receptor kinases (GRKs) and beta-arrestins which perform distinct functions at the receptor [4]. Upon stimulation, GPCRs activate heterotrimeric G proteins. Classically, agonist binding causes a conformational change in a GPCR, inducing guanine exchange factor (GEF) activity that catalyzes the exchange of GTP for GDP on G[alpha] subunits of the heterotrimeric G-protein. This, in turn, leads to the dissociation of the heterotrimeric complex into G[alpha] and G[beta][gamma] subunits. The dissociated subunits promote the formation of second messenger effectors such as cyclic adenosine monophosphate

(cAMP), inositol triphosphate (IP3), diacylglycerol (DAG), as well as modulation of other receptors and channels, such as activation of inward rectifying potassium channels.

1.2 Overview of beta-arrestins

Similar to most biological systems, negative feedback loops have evolved to quench sustained second messenger signaling following receptor stimulation for maintenance of biological homeostasis. After ligand binding and G protein activation, the receptor is phosphorylated on its cytoplasmic loops and C-terminus, primarily by

GRKs [5], which enhance beta-arrestin binding to the receptor. Beta-arrestins were first discovered for their role in mediating receptor desensitization [6], the process whereby repeated stimulation decreases the signaling response over seconds to minutes, through steric hindrance of GPCR interaction with the G proteins. Beta-arrestins also mediate receptor internalization via interactions with clathrin coated pits [7-9]. This can result in downregulation, a sustained decrease in receptor number over minutes to hours due to trafficking of these internalized receptors to proteasomes or lysosomes. Internalized receptors that are not degraded also can be recycled to the plasma membrane [10].

1.2.1 Beta-arrestin signaling roles

It is now established that in addition to acting as negative regulators of G protein signaling, beta-arrestins also couple to numerous signaling mediators including mitogen-activated protein kinases (MAPKs), AKT, SRC, nuclear factor-κB (NF-κB) and phosphoinositide 3-kinase (PI3K) by acting as adaptors and scaffolds [11-18]. These pathways are separate from classical G protein signaling, but can involve similar signaling cascades that are often temporally distinct. More recently, it has also been

appreciated that some receptors tightly interacting with beta-arrestins maintain catalytic

GEF activity on endosomes, continuing to promote G protein signaling after internalization [19-21]. Thus, beta-arrestins regulate nearly all aspects of receptor activity, including desensitization, downregulation, trafficking and signaling.

1.3 A primer on biased signaling

Most drugs that activate or block GPCRs are thought to “equally” target distinct signaling pathways mediated by different G proteins and beta-arrestins. These agonists are thought to amplify downstream signaling pathways in a similar fashion to that of the endogenous reference agonist (“balanced agonists”), while most antagonists are believed to inhibit all second messenger systems activated by those agonists. However, it was appreciated three decades ago that selective agonists or antagonists could specifically target particular receptor-linked effector systems [22]. Indeed, over the past two decades, a number of ligands have been described that selectively activate some pathways while blocking others downstream of the receptor [23, 24]. Compared to the aforementioned balanced agonists, these “functionally selective” [25] or “biased” agonists can selectively activate G proteins while blocking arrestins, or vice versa [26].

This behavior was initially identified in a number of GPCR systems, including PACAP receptor ligands that differentially activated different G proteins as measured by a reversal in potencies [27-29]. Biased agonism has become an increasingly active area of

research since the discovery of beta-arrestin-mediated signaling [30], with a plethora of biased ligands identified for multiple GPCRs [31, 32].

The discovery of biased agonism has had important implications for our understanding of GPCR biology. First, biased agonism is not consistent with two-state models for receptor signaling, and therefore it alters our concept of efficacy [33]. Second, biased signaling suggests that GPCRs should not be modeled as binary switches, but instead as allosteric microprocessors that generate a multitude of conformations in response to different ligands. There are also important clinical implications for these ligands, as selectively activating or inhibiting specific signaling cascades could yield more targeted drugs with reduced side effects [34].

1.3.1 The ternary complex: a validated model for assessing GPCR signaling

While the two-state model for a receptor has been updated, it can be a useful starting point to model aspects of biased signaling. The proportion and relative affinity of two states of the receptor for ligands varies with the intrinsic activity of the agonist and the presence of guanine nucleotides. As described by De Lean, Lefkowitz, and colleagues [35], the simplest model for ligand-receptor interactions involves the interaction of a receptor R with an additional membrane component X, such as a G protein, leading to an agonist-promoted formation of a high affinity ternary complex L-

R-X. Any of the three components of a ternary complex; that is, a ligand, receptor, and transducers, can contribute to a biased response (Figure 1). Ligand bias refers to the 5

situation in which a ligand induces a unique receptor conformation that results in differential coupling to the signal transduction cascade and a biased response. Thus, L[1]-

R-X may be the predominant complex modeled with ligand L[1], activating effector ‘X.’

However, ligand L[2] might produce a distinct complex L[2]-R-Y, leading to activation of effector ‘Y.’

Figure 1: Biased signaling can be encoded through three general mechanisms. (A) balanced agonist binding to a balanced receptor in an unbiased system may display equivalent potencies for two different pathways, such as G protein and beta-arrestin, 6

under assay conditions with similar amplification levels. (B) Biased agonism is encoded through a ligand. A ligand–receptor–effector complex generates distinct conformations that preferentially signal through certain pathways relative to other pathways (beta- arrestin-biased relative to G protein-biased in this example). Unlike the balanced agonist in part a) a beta-arrestin-biased agonist may display a left shift in potency relative to the G protein pathway under the same assay conditions. (C) Biased receptors, such as G protein-biased receptors that lack C-terminal phosphorylation sites necessary for beta- arrestin recruitment, signal preferentially through one pathway relative to another (G protein-biased signaling in this example) despite being stimulated by a balanced agonist. Similar to biased ligands, biased receptors will also display a left shift of one pathway relative to another that may not be observed at an unbiased receptor under the same assay conditions. d) System bias may be due to differential expression of signaling effectors or other cofactors. For example, higher expression of certain G protein-coupled receptor kinase (GRK) and/or beta-arrestin isoforms can bias signaling towards the beta- arrestin pathway (shown here). Alternatively, a lack of GRKs or beta-arrestins can bias signaling towards the G protein pathway (not shown). Emax, maximal effect produced by a ligand.

1.3.2 Modifying components of a ternary complex can bias signaling

Distinct agonists, as well as peptides with disparate post translational modifications, can alter intracellular transduction. For example, either differential glycosylation patterning of the endogenous follicle stimulating hormone (FSH) [36] or different thiazolidinone small molecule allosteric modulators [37, 38] can cause divergent FSH-receptor signaling patterns. Biased agonists can alter the properties of this core ternary complex by binding orthosterically, allosterically, or both in a bitopic fashion. Ligand bias should generate a biased response relatively independent of the cell system tested, although if the transducers required for a biased response are either expressed at low levels or absent, then no change in signaling would be observed.

Biased receptors can be generated by modifying the receptor to change its ability to bind specific ligands or transducers. This can occur through mutation or through 7

differential splicing, both of which can alter coupling to G proteins and beta-arrestins.

For example, all 19 possible amino acid substitutions at Ala293 of the beta-1 adrenergic receptor result in constitutive G protein activity [39], while a single serine to alanine mutation in the C-terminus of the apelin receptor (APJ) inactivates GRK phosphorylation and blunts beta-arrestin signaling [40]. In the C-terminus of neuropeptide Y4 receptor, mutation of glutamic acid, serine or threonine residues disrupts agonist induced recruitment of beta-arrestin-2 and receptor endocytosis [41].

The chemokine receptor CXCR7 was initially classified as a non-signaling ‘decoy’ receptor, although it was later shown that CXCR7 engages ligand dependent beta- arrestin recruitment and signaling while lacking appreciable G-protein signaling [42].

CXCR7 has also been shown to heterodimerize with CXCR4 to alter CXCR4 signaling

[43, 44], while also having distinct and independent functions mediated by beta-arrestin

[45]. The chemokine receptor CXCR3 has splice variants, CXCR3-A and CXCR3-B, that differentially activate beta-arrestins despite only differing in their N-terminal residues, which will be discussed in more detail in chapter 3 [46, 47]. GPCR designer receptors exclusively activated by a designer drug (DREADDs) utilize a chemical-genetic approach for selective activation of a designer GPCR with an otherwise pharmacologically inert compound [48]. Biased DREADDs have recently been designed that selectively activate G protein or beta-arrestin signaling in specific cell types [49, 50].

In addition, biased optogenetic GPCRs for both G protein signaling and beta-arrestin

signaling have been generated, and these chimeric light-sensitive receptors allow for precise temporal and spatial pathway dissection [51, 52].

“System bias”, or “apparent” biased agonism, can be modulated through the differential expression of transducer elements proximal to the receptor, such as the receptor itself, beta-arrestins or GRKs, as well as expression of amplification cascades distal to the receptor [53-55] (Figure 1). Similar to the nuclear hormone receptor system, where a ligand may act as an estrogen receptor agonist in one tissue but as an antagonist in another [56], GPCRs also have different signaling properties depending on the tissue or system probed. Such systems bias can result in different signaling properties depending on the cell type; for example, certain agonists targeting the dopamine-2 receptor (D2R) have different effects in the striatum and prefrontal cortex, which could be related to the differential expression of beta-arrestins and GRKs across brain regions

[57]. System bias may also differ between species [58], and is important to consider in translational studies. Studies of biased ligands with apparently contradictory results may be due to differences in the experimental system. Part of system bias is

“observation bias”, as all measurements are viewed through the lens of a specific assay that is associated with amplification or other properties. To identify ‘druggable’ ligand bias, it is critical to remove the effects of system bias and observation bias from the cellular response [53].

1.3.3 Transmission of ligand bias

How is bias encoded by a ligand transmitted through the receptor to downstream transducers? Our current understanding is that this is primarily accomplished through ligand-induced generation of distinct conformations of the receptor allosteric microprocessor via multiple mechanisms. First, there are changes in receptor secondary and tertiary structure. Then, these conformational changes result in the recruitment of proteins that post-translationally modify the intracellular loops and

C-terminus of the receptor. Together, these changes in conformation and post- translational modifications contribute to differential transducer coupling.

Phosphorylation and ubiquitination of GPCRs, by altering the conformational architecture of the receptor, are the most well described post translational modifications that can bias signaling [59]. GPCRs require phosphorylation of serine and threonine residues within the C-terminus and intracellular loops for tight binding with beta- arrestin. Multiple studies have now demonstrated that differential receptor phosphorylation patterns, or receptor “barcodes” [60-65], lead to distinct receptor conformations or transitions that differentially couple to signaling transducers. The phrase “barcode” was first used by Kim, Lefkowitz, and colleagues in 2005 to describe that the location of phosphorylation sites may constitute such a code that instructs the bound beta-arrestin as to its intended function [60]. For example, stimulation of the beta2-adrenergic receptor (beta-2AR ) with different biased ligands results in discrete

phosphorylation patterns of intracellular residues as assessed by both quantitative phosphoproteomics and antibodies directed against phospho-specific residues [62]. With this receptor “barcode”, biased ligands that activate beta-arrestin signaling induce conformations and trafficking patterns of beta-arrestin that are distinct from those induced by unbiased ligands [62, 66-70].

1.4 Phosphorylation ‘bar codes’ and human disease

Disrupting the aforementioned barcode through mutations in the C-terminus of

GPCRs that remove putative GPCR C-terminal phosphorylation sites can have serious physiological consequences. For example, truncation or mutation of serine or threonine residues in the C-terminus of CXCR4 cause WHIM syndrome by disrupting CXCR4 internalization and sequestering neutrophils within the bone marrow [71, 72]. Mutations that potentiate beta-arrestin-receptor interactions also cause disease. A constitutively active mutation in the conserved E/DRY “ionic lock” motif of the vasopressin 2 receptor

(V2R) leads to constitutive receptor phosphorylation, continual beta-arrestin recruitment and receptor internalization that results in nephrogenic diabetes insipidus through diminished surface expression of V2R and reduced aquaporin channels in renal collecting ducts [73] (Figure 2). Mutating highly conserved polar residues near the transmembrane helical boundaries and core of the GLP1-receptor results in differential regulation of cAMP, calcium, and phospho-ERK signaling and can be a trigger for biased agonism [74-76]. However, it is still largely unclear how these motifs contribute at

the structural level to the transmission of biased information encoded in a ligand to the receptor and then to a transducer.

Figure 2: Drug discovery strategies and physiological consequences of biased signaling. (A) The mu-opioid receptor is a common drug target for analgesia. The endogenous ligand enkephalin (ENK) is unbiased (‘balanced’) with respect to G protein and beta-arrestin signaling. However, current mu-OR -selective agonists, such as morphine, that provide pain relief also cause adverse effects, including respiratory depression, constipation, tolerance and dependence. Both animal and human studies suggest that G protein signaling primarily mediates the analgesic efficacy, while beta- arrestin signaling mediates many of the adverse effects. Strongly G protein-biased agonists, such as TRV130 and PZM21, may therefore provide clinical superiority to currently available agonists. (B) Relative to the wild-type receptor, the naturally occurring human vasopressin 2 receptor (V2R) mutation R137H results in beta-arrestin- biased signaling and is associated with familial nephrogenic diabetes insipidus. The V2R R137H variant is constitutively phosphorylated. This constitutive phosphorylation promotes higher beta-arrestin recruitment and internalization, even in the absence of arginine vasopressin (AVP; also known as antidiuretic hormone), compared with the wild-type receptor. Presumably, a significant reduction in vasopressin-mediated G[alpha]s signaling is sufficient to diminish aquaporin channel insertion into the renal collecting ducts, impeding urine concentration through reduced water resorption and leading to the clinical diagnosis of nephrogenic diabetes insipidus. GRK, G protein- coupled receptor kinase.

1.5 Structural basis of biased agonism

The structure and function of proteins are interwoven. Since the first initial crystal structure of a non-rhodopsin GPCR, the beta-2-adrenergic receptor, was solved in 2007 [77], over 30 high resolution crystal structures of GPCRs have been determined.

This structural revolution has provided invaluable insights into GPCR signaling mechanisms. These structures display a common seven transmembrane architecture, but with significant heterogeneity in the orthosteric ligand binding pockets [78]. Receptors that bind the same ligand have increased conservation within the binding pocket, but still most commonly share only 50-60% of residues [79]. The production of homogenous complexes necessary for crystallization often requires modification of the flexible C- terminus and other intracellular loops; therefore, many GPCR structures lack detailed information of these regions. While active GPCR structures are critical for structure- based drug design for agonists, inactive GPCR structures provide high resolution insight into the development of therapeutic antagonists and inverse agonists. For example, this is true for structures of the angiotensin II type 1 receptor (AT1R) in complex with AT1R blockers (ARBs) [80, 81], a common therapeutic target for anti- hypertensive medications. Inactive structures also provide important insights into signaling mediated by the active state, as a comparison point for conformational differences between ligand bound and unbound receptor states. There are many receptors for which both ‘inactive’ as well either active or partially active intermediate

crystal structures are available, including rhodopsin [82, 83], the A2A receptor (A2AR)

[84-86], the M2 muscarinic receptor (M2R) [87], and the mu-opioid receptor (mOR) [88,

89]. Structures of receptors in complex with biologically potent allosteric antagonists or negative allosteric modulators include CC Chemokine receptor 2 (CCR2) [90], CC

Chemokine receptor 9 (CCR9) [91], corticotrophin releasing factor receptor (CRHR1)

[92], and the glucagon receptor [93]. These offer high resolution insight into druggable surfaces outside the orthosteric ligand pocket, most notably rearrangements of amino acid contacts between transmembrane regions 3, 6, and 7 that expose residues that could lead to GEF activity and subsequent G protein activation. However, the changes observed in these structures can be quite limited.

These structures have shown that agonists alone are often not sufficient to stabilize an active GPCR conformation. Transducer coupling, or use of a stabilizing modulator such as a nanobody, is often required for trapping the receptor in its active state. As of the end of 2017, only three GPCR structures have been solved (and published) in complex with a bona fide G protein [94-96]. The beta-2AR was the first such receptor-G protein complex, and the co-crystalization structure reveals that both the N- terminal and C-terminal domains of the G[alpha]s subunit interact with the beta-2AR intracellular loop 2, transmembrane domain 5 (TM5), and TM6. Recently, the structure of the calcitonin receptor (CTR) in complex with a G[alpha]s heterotrimeric complex was solved by cryo-EM [95]. Like the beta-2AR, significant outward movement of TM6 is

observed. Despite the beta-2AR belonging to the class A GPCR sub-family and the CTR belonging to the class B sub-family (comprising of a larger extracellular N-terminal domain when compared with class A GPCRs), an overlay of the beta-2AR and the CTR

G protein complex structures reveal only minor differences in G protein conformation between the two receptors. However, in contrast to the beta-2AR, helix VIII of the CTR appears to play a more important role in receptor stability at the cell surface and in interactions with G protein. Indeed, growing evidence suggests an important role of helix VIII in signaling. At the enigmatic angiotensin II type 2 receptor (AT2R), helix VIII lies parallel to the membrane in the active-like state apparently sterically inhibiting G protein and beta-arrestin interactions with the AT2R, which is in agreement with an unusual lack of observed G protein or beta-arrestin signaling for this receptor [97].

Other GPCRs have been crystalized with G-protein “mimics,” such as nanobodies or an engineered mini-G protein model, that hold the receptor in a conformation thought to mirror the G protein activated state. These structures include the M2 muscarinic [87], the mOR [89], the viral US28 receptor [98], and the adenosine

A2A receptor (A2AR) [99]. Two distinct conformations of the beta-2AR stabilized by different nanobodies have also been recently reported [100].

Structural information on how beta-arrestins can interact with GPCRs is also beginning to emerge. Truncation of the arrestin-1 (visual arrestin) C-terminus mimics an activated arrestin, and releases a critical central-crest ‘finger loop’ by disrupting the

polar core [101]. The structure of this finger loop bound to rhodopsin, as well as of the structure of rhodopsin bound to arrestin1, were recently solved, providing a high resolution view of an additional GPCR:transducer complex [102, 103]. Additional structures of GPCRs in complex with beta-arrestins, as well as GRKs, will be necessary to determine if conserved receptor:arrestin interaction patterns are also present. Such studies will reveal if G proteins, GRKs, and arrestins have preferences of specific receptor conformations. Comparing structures of the ligand:receptor:transducer ternary activated complex to the inactive receptor state are the cartographical methods of rational GPCR drug design, and represent a powerful tool beginning to be harnessed for increasing the efficiency of drug discovery.

1.6 Emerging strategies for determining GPCR structure

A range of other biophysical studies are now being used to provide high resolution information to enable drug discovery for more challenging targets [104].

Electron microscopy techniques (EM), notably cryo-EM and negative stain EM, have emerged as important methods for determining GPCR-transducer structures. An advantage of EM technology is the ability to study a receptor in its wild-type form, as x- ray crystallography frequently requires receptor modifications to obtain sufficient homogeneity and stability for crystal formation. In addition to high resolution structures such as the CTR-G protein complex described earlier, low resolution structures from negative stain EM have been used to reveal agonist-occupied beta-2AR in complex with

G protein as well as to capture transient intermediate G protein and beta-2AR complexes [105]. Furthermore, EM of a beta-2AR and vasopressin type 2 receptor (V2R) chimera receptor (utilized to increase beta-arrestin affinity) demonstrates that beta- arrestin can adopt at least two distinct conformational states when interacting with a

GPCR. One conformation reveals beta-arrestin bound only to the phosphorylated C- terminus of the receptor, while a separate conformation demonstrates the flexible

“finger loop” of beta-arrestin inserting into the receptor transmembrane core in addition to the C-terminus interaction [106]. The functional significance these distinct beta- arrestin receptor interactions (C-terminus only and C-terminus+core) has also been studied. Partial engagement of beta-arrestin with the C-terminus appears sufficient for receptor endocytosis and ERK activation [107]. Contact of the beta-arrestin finger loop with the receptor transmembrane core appears necessary for desensitization, with negative stain EM providing support that a finger loop-core interaction sterically blocks the apparent G protein binding site [108]. Negative stain EM studies have also revealed that internalized receptor complexes can consist of a GPCR, beta-arrestin, and G protein

[109]. Such internalized ‘megaplexes’ demonstrate that a single GPCR can simultaneously interact with both a beta-arrestin and a G-protein. Visualization of these

‘megaplexes’ provides a molecular basis for sustained GPCR catalytic GEF activity and subsequent G protein signaling following GPCR internalization into endosomes. Taken

together, the existing EM studies lend further structural support for the presence of multiple GPCR ‘active’ conformational states.

Additional experimental approaches, including a high-resolution mass spectrometry labeling strategy, NMR, molecular dynamic simulations, conformationally selective RNA aptamers, and single domain camelid antibodies (nanobodies) [100, 110-

112], have shown that functionally similar ligands can induce distinct receptor conformational changes. For example, 19F NMR spectroscopic analysis of the beta-2AR demonstrated distinct receptor conformational states when bound to balanced or biased ligands. The FDA approved beta-blocker Carvedilol, proven to be particularly effective for the treatment of heart failure, is a moderately beta-arrestin-biased agonist at the beta-

1AR [113, 114] and beta-2AR [115]. A comparison of beta-2AR conformations induced by either the balanced agonist isoproterenol or the beta-arrestin-biased agonist carvedilol revealed that that G protein activity correlates with movement of transmembrane helix

VI, whereas carvedilol chiefly alters the conformation of helix VII [116]. RNA aptamer binding also revealed specific beta-2AR conformations induced by different ligands

[117], and 13C-labeled beta-2AR s further validate distinct receptor states not captured by previous crystal structures [118]. Double electron electron resonance (DEER) spectroscopy of a trifluoro-methyl labeled cysteine at TM6 of the beta-2AR has also shown significant conformational heterogeneity and rapid interconversion of multiple receptor states [119]. In addition to the beta-2AR, a multitude of active receptor

conformations are observed at the ghrelin [120] and serotonin 2B [121] receptors, demonstrating that a heterogeneous ensemble of active receptor states is a conserved property. Beta-arrestin biosensors have also recently been employed to correlate the conformational signature of arrestins to predict signaling and trafficking functions following drug stimulation [69, 70]. Beta-arrestin NMR probes show different beta- arrestin-phosphopeptide interactions encode distinct structures that correlate with specific beta-arrestin mediated functions [122]. In summary, most structural studies support a model of the receptor as an allosteric microprocessor, with biased ligands inducing an array of distinct receptor conformations that differentially recruit and activate transducers.

1.7 Beta-arrestins

Beta-arrestins are ubiquitously expressed proteins that were first described for their role in desensitizing G protein-coupled receptors (GPCRs) [6]. We now appreciate that these proteins are multifunctional adapter proteins that regulate a vast array of cellular functions. As beta-arrestins, along with G proteins, are appreciated to direct distinct signaling pathways, they provide validated intracellular effectors to qualitatively and quantitatively assess bias. This section will provide background into this critical protein family, which regulates a diversity of cellular signaling throughout every of our biological systems.

Beta-arrestins were identified through their sequence homology to visual arrestin

(arrestin-1), so named because of its ability to “arrest” rhodopsin signaling in the retina.

There are two beta-arrestin isoforms, beta-arrestin1 and beta-arrestin-2 (also denoted as arrestin-2 and arrestin-3, respectively). Both are expressed ubiquitously and share 78% sequence homology [123]. Beta-arrestins are highly conserved across species, with ~50% sequence homology between vertebrates and invertebrates. The other arrestins are expressed in the eye: arrestin-1 (visual arrestin) and arrestin-4 (cone arrestin) [124].

There are other proteins, termed beta-arrestins or arrestin-domain containing proteins, that share the arrestin structural fold and are involved in receptor endocytosis, although the full breadth of their functions is still emerging [125]. Similar to arrestin’s function in the visual system, beta-arrestins were first identified for their capacity to desensitize beta-2 adrenergic receptor (beta-2AR) G-protein signaling following agonist stimulation

[6]. Through a number of investigations, it became apparent that the two beta-arrestins isoforms shared the capability to interact with activated GPCRs, but that they differed in terms of their expression patterns, their specificity for different GPCRs, and their functional effects [126]. We now appreciate that the beta-arrestins regulate a diverse array of cellular processes including MAPK (Mitogen-Activated Protein Kinase) signaling, receptor transactivation, receptor trafficking, and transcriptional regulation in addition to the canonical roles of GPCR desensitization and internalization [127, 128].

These studies have revealed the current spectrum of beta-arrestin-mediated cell processes downstream of GPCRs (Figure 1).

1.7.1 Distinct and Overlapping Roles for the beta-arrestins

Beta-arrestin-1 and beta-arrestin-2 knockouts are phenotypically normal and produce viable progeny, but these mice display abnormal responses to physiologic stresses [129, 130]. Notably, the double knockout of beta-arrestin-1 and beta-arrestin-2 in mice is perinatal lethal, strongly suggesting a compensatory ability for each isoform

[131]. Nevertheless, important differences between beta-arrestin isoforms are present.

While both accumulate in the cytoplasm following overexpression, beta-arrestin-1, but not beta-arrestin-2, accumulates in the nucleus. While both beta-arrestin-1 (418 aa) and beta-arrestin-2 (410 aa) have nuclear localization sequences on their N-termini, only beta-arrestin-2 has a nuclear export sequence located on its C-terminus, thereby accounting for differences in nucleocytoplasmic shuttling [132]. Beta-arrestin-1 and -2 scaffold to different signaling pathways; however, this is often cell type- and receptor- specific. Beta-arrestin-2, but not beta-arrestin-1, is known to be necessary for creating a signaling that activates c-Jun amino-terminal kinases (JNKs) [133]. Beta-arrestin-1 and beta-arrestin-2 can “reciprocally regulate” signaling at certain receptors; that is, one isoform increases pathway specific signaling while the other isoform inhibits signaling.

Reciprocal regulation is observed in the type 1 Angiotensin II receptor (AT1R), where siRNA knockdown of beta-arrestin-2 attenuates ERK signaling while knockdown of

beta-arrestin-1 potentiates ERK signaling [134]. However, at other receptors such as the beta-2 adrenergic receptor and the type 1 parathyroid hormone receptor (PTH1R), knockdown of either beta-arrestin-1 or beta-arrestin-2 decreases ERK signaling [12, 135].

In addition, growing evidence suggests that the kinetics of beta-arrestin-mediated signaling is tissue dependent [136]. Adding to the complexity, the function of beta- arrestins appear to be strongly influenced by its cellular environment, such as the presence or absence of critical signaling partners such as GRKs [137].

1.7.2 Beta-arrestin post-translational modifications

Post-translational modifications are critical to beta-arrestin signaling and trafficking. Beta-arrestin-1 and beta-arrestin-2 are constitutively phosphorylated and both require C-terminal dephosphorylation for targeting internalized receptors to clathrin. The phosphorylation site differs between beta-arrestin isoforms (Ser 412 for beta-arrestin-1, Ser 361 and Thr 383 for beta-arrestin-2) [138]. However, dephosphorylation of beta-arrestins is not required for desensitization of G protein signaling. Differential trafficking of beta-arrestin isoforms often control the kinetics of desensitization, and if or when a receptor is recycled back to the membrane surface.

Dephosphorylation of beta-arrestins following receptor activation is necessary for full functionality, including receptor internalization and beta-arrestin-mediated MAPK signaling. Covalent modification of beta-arrestin with ubiquitin (ubiquitination) results in sustained beta-arrestin:GPCR complexes and prolonged MAPK activity.

Ubiquitination of a GPCR is necessary for receptor degradation, and ubiquitination of beta-arrestins is necessary for GPCR internalization. Different patterns of beta-arrestin ubiquitination (especially at Lys11 and Lys12) result in changes in receptor trafficking

[139] and the ability to scaffold signalosomes [140]. Other reported modifications that regulate beta-arrestin function are S-nitrosylation [141] and SUMOylation [142], and it is likely that beta-arrestins are modified in other, yet unexplored, ways that impact their functions.

1.7.3 Beta-arrestin-regulated Desensitization

Receptor desensitization is the process by which repeated stimulation of a GPCR results in a decreased response over seconds to minutes. This is in contrast to downregulation, the process underlying decreased signaling that occurs over hours.

Receptor-dependent activation of heterotrimeric G proteins induces dissociation of

G[alpha] and G[beta][gamma] subunits, promoting their interactions with effector proteins that lead to downstream signaling. Desensitization of GPCR signaling requires a coordinated response by G-protein receptor kinases (GRKs) and beta-arrestins [143].

The first functional effect noted in the arrestin family was the desensitization of G protein-mediated signaling by rhodopsin [144]. G protein signaling inhibition was soon recognized as a function of beta-arrestins in tissues outside of the visual system, and inhibition of G protein-mediated signaling was the primary function assigned to beta- arrestins until the mid-1990s. Beta-arrestins are thought to quench G-protein signaling

by sterically inhibiting the G-protein interaction at the second (ICL2) and third (ICL3) intracellular loops of a GPCR [126, 145, 146]. This steric hindrance uncouples GPCRs from the G-protein signal transduction process, which results in desensitization of second messenger pathways [147].

Phosphorylation of the cytoplasmic elements of GPCRs is critical for beta-arrestin recruitment and receptor desensitization [5, 143, 144, 148]. GPCR phosphorylation can be targeted directly to intracellular regions of the ligand bound receptor complex

(homologous desensitization) or to multiple GPCRs throughout the cell (heterologous desensitization). Heterologous desensitization is often mediated by protein kinases A

(PKA) or C (PKC) [149]. In homologous desensitization, phosphorylation of the GPCR intracellular residues is predominately mediated by G-protein receptor kinases (GRKs)

[5]. There are seven GRK isoforms: GRK1 and 7 are confined to the visual system, GRK2,

3, 5, and 6 are ubiquitously expressed, and GRK4 is expressed primarily in the reproductive tract [150]. Importantly, phosphorylation of GPCRs appears to be absolutely required for desensitization. Elimination of intracellular phosphorylation, either by using phosphorylation-deficient receptor mutants or by co-transfecting a dominant-negative GRK, abolishes beta-arrestin recruitment, desensitization and internalization [148, 151, 152]. This process occurs sequentially, as beta-arrestin binding requires both ligand-induced conformational change in the GPCR and GPCR phosphorylation [153]. Since GRK-mediated phosphorylation of receptors is often the

rate limiting aspect of receptor desensitization, it can dominate the kinetics of beta- arrestin binding to receptors in intact cells. Heterogeneity in the phosphorylation sites is a second source of complexity, since GRK-mediated phosphorylation occurs not only at the C-terminal tail of the receptor (e.g., rhodopsin and the beta-2AR) but also at many other intracellular sites, most notably ICL3 (e.g., beta-2 adrenergic receptor [154] and M2 muscarinic receptor [155]).

1.7.4 Beta-arrestin-regulated Receptor Trafficking

For a number of GPCRs, beta-arrestins function as adapters to target receptors to clathrin coated pits through its scaffolding of AP-2 and clathrin [156]. Many, but not all

[157-159], GPCRs appear to require beta-arrestin for internalization. The recruitment of beta-arrestin-2 to receptors can be modified by mutation of selected “receptor discriminator” residues [160]. Receptors that follow the clathrin-dependent endocytic pathway are internalized in clathrin-coated pits in a dynamin-dependent fashion [161].

Beta-arrestins scaffold multiple protein regulators including ARF6 [162] and n- ethylmaleimide-sensitive fusion protein [163], which are implicated in beta-arrestin- mediated receptor internalization. Once internalized, the receptor continues to tubulovesicular early endosomes. Here, receptors are sorted to either recycling endosomes, which return GPCRs to the plasma membrane, or multivesicular late endosomes, which traffic receptors to lysosomes for degradation [127]. Some GPCRs internalize in the absence of beta-arrestins, but require them for recycling [164].

GPCRs that traffic through the clathrin- dependent endocytic pathway can be divided into two groups, class A and B, based on the characteristics of agonist- dependent beta-arrestin binding [165]. Beta-arrestins facilitate the desensitization and internalization of both receptor classes. Class A receptors, such as the beta-2-adrenergic receptor, bind beta-arrestin-2 with greater affinity than beta-arrestin-1. Class B receptors, such as the V2 vasopressin receptor, bind beta-arrestin-2 and beta-arrestin-1 with approximately equal affinities. In class A interactions, receptors internalized in membrane vesicles remain at the cellular surface, and beta-arrestins dissociate from the receptor at or near the plasma membrane. In class B interactions, beta-arrestins form a long-lived complex with the receptor and traffic into endosomes. Class A receptors are associated with transient beta-arrestin ubiquitination and class B receptors with stable beta-arrestin ubiquitination. Notably, class A patterns can be switched to class B by covalently linking ubiquitin to beta-arrestin or by switching the C-terminus of the receptor to that of a class B receptor [139]. Differential phosphorylation by GRKs and other kinases also regulate the receptor-beta-arrestin interaction [55]. These changes in receptor and beta-arrestin post-translational modifications appear to be ligand dependent, as different ligands binding to the same receptor can result in class A or B patterns. In addition to ligand stimulated receptors, constitutively activate receptors that internalize in the absence of ligand appear to rely on beta-arrestins for trafficking [73].

Beta-arrestins can also traffic receptors to distinct areas of the cell, such as beta-arrestin

translocation of Smoothened (Smo) during hedgehog pathway activation. Once formed, this beta-arrestin:Smo complex localizes to the primary cilia, where the complex activates Gli transcription factors [166].

1.7.5 Beta-arrestin-regulated receptor signaling

It is now appreciated that in addition to regulating receptor-stimulated G protein signaling, beta-arrestins are also capable of initiating distinct signaling patterns [14].

These signaling patterns are often both spatially and temporally distinct from G protein- mediated signaling, and result in unique cellular, physiological and pathophysiological consequences. In addition to differential trafficking, beta-arrestins also scaffold MAPKs, including ERK1/2. Both G proteins and beta-arrestins mediate ERK1/2 activation, but through distinct mechanisms. Recruitment of beta-arrestins sterically inhibits G protein interaction with the active receptor, thus quenching the rapid G protein mediated phase of ERK activation. Sometimes G protein-mediated ERK activation can also include a slow phase [167], so kinetics alone cannot distinguish between G protein- and beta- arrestin-mediated ERK signaling. Separately, beta-arrestin scaffolds Raf-1, MEK1, and

ERK, thus serving to sequester ERK in the cytosol [168]. Seclusion of phosphorylated

ERK1/2 in the cytosol precludes ERK-mediated transcription and prolongs ERK signaling. Similarly, beta-arrestin-2 scaffolds JNK1/2 with its upstream kinases MKK4 and MKK7, which phosphorylate different residues in its activation loop [133].

Activation of p38 signaling cascades is also beta-arrestin-dependent, although a direct

scaffolding complex of beta-arrestin and p38 has not been elucidated [169, 170].

Modified beta-arrestins have also been reported to signal to kinases independently of

GPCRs [171].

Ubiquitination is now appreciated to not only regulate protein degradation, but also protein signaling. In addition to being ubiquitinated itself, beta-arrestins act as adapters for multiple E3 ubiquitin ligases. Complexes containing beta-arrestin and E3 ligases are essential for mediating aspects of ubiquitin-dependent signaling. For example, beta-arrestins are critically involved in ubiquitination of receptors, acting on late endosomal populations as a lysosomal degradation signal for the receptor. More broadly, beta-arrestins act as adapters for several E3 ligases that catalyze ubiquitination, such as Mdm2. Mdm2 ubiquitination of beta-arrestin-2 is required for clathrin-mediated internalization of the beta-2AR [172], while the E3 ligase AIP4 is necessary for sorting of

CXCR4 to early endosomes and then lysosomes [173]. Endosomal sorting of CXCR4 also require beta-arrestin-1 interaction with STAM-1, part of the endosomal sorting complex required for transport (ESCRT-0) machinery [174]. Beta-arrestins are also regulated by deubiquitinating enzymes such as the ubiquitin specific protease USP33 [175, 176] thus providing a mechanism for coordinating receptor recycling and resensitization.

Interestingly, evidence suggest that receptor posttranslational modification can influence later signaling events, either by catalyzing additional posttranslational modifications or by controlling downstream signaling pathways [173, 177].

A number of other signaling pathways have been demonstrated to be regulated by beta-arrestins. The transactivation of epidermal growth factor (EGF) receptor by

GPCRs can be regulated by beta-arrestins, through the activation of a transmembrane matrix metalloprotease that cleaves membrane-bound EGF ligand [178]. Beta-arrestin-2 can inhibit NF-kB signaling through stabilization of IkBa [11]. Beta-arrestin-1 can directly influence epigenetic modifications through nuclear interaction with histone acetylases and deacetylases that regulate chromatin structure [179]. There are now even examples of beta-arrestin-mediated G protein signaling. Beta-arrestins promote G protein signaling by the type 1 parathyroid hormone [180] and V2 vasopressin receptor

[181] from endosomes, an effect that is lost with beta-arrestin knockdown. The beta-2AR has also been noted to maintain an active conformation that can signal through G proteins to generate cAMP from endosomes [21]. These finding suggests that beta- arrestin trafficking of receptors to endosomes results in a receptor that is still capable of activating G proteins. This signaling appears to be mediated by a complex of receptor: beta-arrestin:G protein [180], direct evidence of which would fully overturn the classic paradigm of beta-arrestins as “arresting” G protein signaling.

1.7.6 Beta-arrestin-biased agonism

Following the discovery of beta-arrestin-mediated signaling, came the observation that some ligands are capable of selectively signaling through beta-arrestins while blocking signaling through G proteins. This, as mentioned previously, is an

example of biased agonism, which is the ability of certain agonists to signal through different pathways of a GPCR with different efficacies. Strongly biased agonists activate one pathway while completely blocking signaling through others, while partially biased agonists may strongly signal through one pathway while weakly signaling through another. Biased agonism between different G proteins has been appreciated for 30 years

[182] and the discovery of beta-arrestin-biased agonism resulted in renewed interest in this area [34]. Such bias towards beta-arrestin or G proteins implies differential recruitment of GRKs, as receptor phosphorylation by GRKs is required for sustained beta-arrestin coupling. Biased agonism changes the classical models of receptor theory associated with single active and inactive receptor conformations to one with multiple receptor conformations. While balanced ligands stabilize the conformations that are competent for signaling to all downstream pathways, biased ligands stabilize only those conformations that are capable of promoting a subset of downstream signaling effectors.

For example, ligands can show bias for either G protein- (G protein-biased) or beta- arrestin-mediated (beta-arrestin-biased) signaling. This is necessarily an oversimplification, as recruitment of beta-arrestins requires recruitment of additional signaling components, such as GRKs.

Such bias adds a layer of complexity to the traditional definition of ligand action.

For example, a beta-arrestin-biased AT1R agonist has markedly different physiologic effects from the endogenous agonist angiotensin II (AngII) [183]. While AngII causes

vasoconstriction, cardiac hypertrophy and increased cardiac contractility, the beta- arrestin-biased agonist causes vasodilation, no cardiac hypertrophy but still increases cardiac contractility via beta-arrestin-mediated phosphorylation of tropomyosin and other contractile proteins [184]. A number of other G protein- and beta-arrestin-biased agonists targeting a variety of receptors are currently being tested in early phase clinical trials, including those of the mu-opioid receptor [185] and apelin receptor [186].

While many biased agonists have been identified serendipitously, drug development of biased agonists requires an approach for quantifying the degree of ligand bias. Classical parameters of receptor signaling such as maximal effects (Emax) and potencies (EC50) cannot account for differences in receptor reserve and amplification of different signaling pathways [187]. In assays with significant amplification, such as second messenger assays, e.g., cyclic AMP formation, both full and partial agonists can reach the same maximal response, while in assays with little amplification, such as assays that monitor recruitment of beta-arrestin to a receptor, partial agonists have significantly lower maximal responses than full agonists. Multiple approaches have been developed that all address the issue of differential amplification between signaling assays [187-189]. As an example, bias factors [187] yield an estimate of bias equivalent to other approaches, and when combined with dissociation constants obtained from a binding experiment, also provide an estimate of relative efficacy. All of these approaches

yield similar estimates for bias [190], although relative errors can be significantly higher depending on the assumptions made in the analysis [53].

1.7.7 The signaling barcode: A model for allosteric regulation of beta- arrestins

Numerous studies have suggested that beta-arrestins can adopt multiple conformations that differentially regulate distinct cellular signaling events. Regulation of these unique beta-arrestin conformations is controlled at a number of levels, through interactions with the ligand:receptor complex, different post translational modifications of both the receptor and of beta-arrestin, and the presence of other cofactors that are cell type-dependent. As mentioned previously, these different mechanisms for beta-arrestin regulation have been integrated in the “signaling barcode” model for receptor [136].

Binding of beta-arrestin to distinct receptor C-terminal phosphorylation patterns

(“barcodes”) generated by different kinases, results in different conformation of receptor-bound beta-arrestin. These different beta-arrestin conformations are capable of activating distinct downstream signaling events, such as endocytosis, desensitization or kinase activation. While an attractive hypothesis, there is still only limited data to support it. At the M3 muscarinic receptor, differential phosphorylation of the receptor

C-terminus was noted in response to different ligands and in different tissues

(presumably due to differential expression of GRKs and other kinases) [63]. At CXCR4, unique serines are phosphorylated by PKA, GRK2 and GRK6, with different effects on

ERK1/2 phosphorylation and calcium influx [61]. At the beta-2AR, the best studied of 32

these receptors, a beta-arrestin-biased ligand resulted in phosphorylation of distinct sites by GRK2 and GRK6 compared to a balanced agonist, with different effects on receptor endocytosis and signaling through MAP kinases [62]. Important questions that need to be addressed in further developing the barcode model are how differential recruitment of GRKs to the receptor influences receptor phosphorylation, how the receptor allosterically induces conformational changes in the structures of beta-arrestin, and the means by which specific post-translational modifications of beta-arrestins directly influence their conformation and subsequent downstream signaling.

1.7.8 Signal transduction to effectors

Recent structural studies have addressed the question of how beta-arrestins transduce signal encoded in the receptor to effector molecules. The beta-arrestins can interact with downstream effectors in different ways; for example, in beta-arrestin-1, an intrinsically disordered clathrin binding box interacts with clathrin between blades 1 and 2 of its beta-propeller, or via an 8-amino acid splice loop found only in its long isoform that interacts with a binding pocket formed by blades 4 and 5 [191]. Further insights into the allosteric regulation of beta-arrestin signaling has recently been provided by an NMR study that used 19F probes in beta-arrestin-1 to probe changes in its structure induced by different phosphopeptides derived from the V2R [122]. While all the phosphopeptides interacted with the phosphate sensor to induce changes in the finger and middle loops, there were also distinct phospho-interaction patterns that were

related to the spacing of the multiple beta-arrestin phospho-binding sites. These distinct patterns may serve as a structural model for the signaling barcode, by which changes in a GPCR phosphorylation pattern are translated to distinct conformations of beta-arrestin that can be “read” by downstream effectors.

2. Quantifying Bias

2.1 Historical context of quantifying receptor signalling

In pharmacological models of receptor signaling, such as those of Ariens [192],

Stephenson [193], Furchgott [194], and Black and Leff [195], a ligand is considered to have two primary properties in interactions with a receptor: affinity (the ability of a ligand to form a ligand–receptor complex) and efficacy (the ability of the ligand– receptor complex to increase or decrease downstream signaling response(s)). In mechanistic models of receptor signaling, such as the ternary complex model, efficacy is a measure of the ability of a ligand to effect the transition between active and inactive receptor conformations (such as the alpha parameter in the ternary complex model), while in pharmacological models, efficacy relates the pharmacological stimulus to the observed response [193]. These mechanistic and pharmacological estimates of efficacy are closely related mathematically [53]. As noted above, the concept of biased agonism requires that a receptor display multiple efficacies and determination of these biased efficacies requires a deconvolution of ligand bias from system bias. Multiple approaches have now been proposed to quantify ligand bias [187, 188, 196], although identifying

biased ligands requires multiple steps, from the choice of assays used to assess different signaling pathways to the computational approaches used to quantify ligand bias.

2.1.1 Assays for detecting G protein and beta-arrestin signaling

Table 1 provides a summary of different experimental techniques that can be utilized to measure signal transduction effects. Broadly speaking, these assays are based on different aspects of transducer activity, which include transducer redistribution, receptor:transducer proximity, transducer conformation, receptor internalization, and transducer signaling. Redistribution assays are based on the movement of transducer either to or away from the receptor or downstream effector with ligand stimulation.

Receptor proximity assays quantify changes in the distance of a population of transducers, such as beta-arrestins, to a GPCR upon agonist binding. Assays of transducer conformation and signaling both require a clear understanding of the relationship between the measured effect and transducer activity. For example, while beta-arrestin activation is associated with conformational changes, only certain conformational signatures are associated with specific signaling function, such as receptor internalization [69]. Indeed, receptor internalization does not always require beta-arrestin recruitment, or vice-versa [157]. Some signaling assays may have inputs from multiple upstream transducers, such as MAP kinase phosphorylation, which has distinct inputs from both G proteins and beta-arrestins.

Table 1: Selected assays for assessing ligand bias

Assay Technology Strengths Weaknesses

Proximity Intermolecular -Sensitive -The transducer BRET or FRET -No amplification and/or receptor must between the -Easy to transiently be modified receptor and transfect the tagged -Depending on the

beta-arrestin receptor and timescale resolution, pertinent tagged may only detect effector into certain stable interactions cell lines -Overexpression levels may not represent actual expression in the tissue of interest -G protein assays usually require

BRET2, which is more technically challenging

Enzyme -Sensitive -Amplification may

complementation -Kits available for overestimate Emax (for example, quick screening -Depending on the cAMP-dependent Reporter-based timescale resolution, firefly luciferase) may only detect stable interactions -Many assays involve modification of either 36

the receptor or transducer

Reporter-based -Very sensitive -Amplification may

assays -High throughput overestimate Emax -Open source -For beta-arrestin resources available assays, beta-arrestin for the majority of and/or the receptor non-olfactory human are usually modified

GPCRs (PRESTO- Tango)

Conformation Intramolecular -Broad insight into -Difficult BRET or FRET of structural changes interpretation, must beta-arrestin [67, -New generation of control for avidity 69] or seven biosensors with -Requires transmembrane multiple modification of receptor donor/acceptor sites receptor or biosensors [197] for greater resolution transducer

RNA aptamers -Molecularly diverse, -Requires significant [117] allowing for increased screening and conformational enrichment to granularity identify receptor- specific aptamers

Redistribution -Labeling of beta- -Potential to work -May miss transient arrestin or with native receptor, beta-arrestin receptor [198] (e.g. fluorescently interactions labeled beta-arrestin2- GFP recruitment) -Easy to apply to 37

transiently transfected cell lines -Labeling of -Can utilize native -May require strong receptor / receptor (if performed antibody to receptor receptor with antibody) or -Only measures total internalization modified receptor receptor with a tag internalization, not -Flexibility with cells beta-arrestin utilized dependent internalization Signaling -Multiple -Directly tests a -Often difficult to outputs, functional response; conduct in a high including potentially more throughput manner phosphorylation physiologically -Must control for off- (e.g. MAPK 1/2, relevant target effects (e.g. AKT), native receptors in a ubiquitination, cell line) chemotaxis, apoptosis, stress fibre formation -TGF alpha -Granular -Requires specialized ectodomain quantification of G cell line shedding assay protein isoforms -Not all G protein [199] -High throughput isoforms are potential compatible

Cyclase -High throughput -Must control for off accumulation -Sensitive target effects -Compounds that permeate the cell can cause false positives by directly altering adenylyl cyclase activity Calcium/potassiu -High throughput -Dyes can diffuse out m indicator dyes -Sensitive of the cell, reducing -Some dyes can be signal targeted to specific -Must control for off cellular domains target effects

Calcium influx -High throughput -Coelenterazine is (Aequorin) -Sensitive irreversibly -Aqueorins can be consumed targeted to specific -Must control for off organelle for cellular target effects compartment analysis -Compounds that -Unlike calcium permeate the cell can sensitive dyes, large cause false positives size of aequorin prevents cellular diffusion

When designing experiments to identify biased agonists, it is important to eliminate sources of system bias that can confound data interpretation. Comparisons of efficacies and potencies from assays are often limited by differences in receptor reserve

(also known as ‘spare receptors’) or amplification between assays. In assays with significant amplification, such as G protein second-messenger assays, both full and partial agonists can reach the same maximal response. In assays with negligible signal amplification, such as BRET-based recruitment or dissociation assays, a balanced partial agonist will display less efficacy relative to a full agonist. With a simple comparison of potencies or maximal responses, a partial balanced agonist that yields half maximal response in assay B and a maximal response in the amplified assay A could incorrectly be labeled as a biased ligand relative to the full balanced agonist that that achieved maximal responses in both assays. Potencies are also affected by amplification. In assays with high amplification, a full agonist will have a greater leftward shift in potency (EC50) from its dissociation constant (KD) than a partial agonist. However, in assays with no or

minimal receptor reserve, the EC50 will approximate the KD for a partial agonist, but not for a full agonist. It can be difficult to compare proximal and distal signaling effectors due to varied amplification and system bias [200]. Thus, it is not straightforward to compare Emax and EC50 alone to identify biased agonists. To address this issue, both qualitative and quantitative approaches have been developed to attempt to remove the effects of system bias and identify truly biased ligands.

2.1.2 Identifying Biased Ligands Qualitative assessment

To increase the sensitivity and specificity of identifying biased compounds, both quantitative and qualitative methods should be used to identify potentially biased ligands. The effects of system bias can be assessed qualitatively by a ‘bias plot’ [201]

(Figure 3). Bias plots are generated by converting dose-response data for two signaling pathways of interest, e.g., G protein and beta-arrestin, into plots comparing responses in pathway A to responses in pathway B at identical concentrations of ligand [200, 201].

Figure 3: General approach to characterizing biased ligands. First, assays for different pathways should be chosen with the goal of minimizing differences in signal amplification. Such assay selection optimizes the window for identifying biased agonists (see example of a bias plot). Time-dependent and cell-dependent data should be obtained to ensure that there are no significant kinetic or cell-specific effects. To qualitatively identify biased agonists, construct a ‘bias plot’ by, for example, graphing beta-arrestin activity on the x axis and G protein activity on the y axis (each normalized to pathway-specific maximal signals in this example). Deviations from the reference agonist suggest the presence of ligand bias. If no biased signaling is observed on a bias plot, then it is unlikely that true biased signaling is present (even if calculated bias factors are statistically significant). Multiple approaches can be used to calculate bias factors, such as a method based on intrinsic relative activities (calculator available online). After identification and quantification of ligand bias, the physiological implications of such signaling can be tested in relevant cell lines or primary cells. Biased signaling properties can then be confirmed in the receptor from a relevant model organism and subsequently evaluated in animal models for safety and efficacy.

An advantage of the bias plot is it allows an assessment of assay amplification effects that can confound efforts to identify biased ligands. It also provides a way to identify the

best assays to quantify ligand bias, as it provides information on the windows for identifying G protein- or beta-arrestin-biased agonists. When choosing assays to assess ligand bias, it is important to select assays that have similar levels of amplification.

Similar levels of amplification provide the largest windows for identifying ligands that could be biased towards either pathway tested. However, if one is interested in identifying ligands biased towards pathway A relative to pathway B, it can make sense to choose an assay with somewhat more amplification in pathway B relative to A. If a ligand does not appear to be biased using a bias plot, it is unlikely to be biased, even if a

“bias factor” from a quantitative approach is significant. This is because the bias plot is not prone to errors introduced from different fitting approaches [53]. However, even bias plots cannot account for other aspects of system bias, such as differential expression of GRKs and beta-arrestins, which can qualitatively change the relative difference between downstream pathways.

2.2 Translating in vitro bias into in vivo utility

Many of the strategies used to bring a promising biased ligand from the bench to bedside are similar to those for typical drug candidates and have been reviewed elsewhere [202-205]. Given the significant costs in late phase drug discovery, accurately quantifying the relative signaling properties of biased agonists early in the drug discovery process, as well as evaluating effects in suitable preclinical models of disease, is necessary. A number of approaches for biased ligand drug discovery can build

confidence that the observed physiological effects are due to bias at the intended target and pathway (Figure 3). Often, the first step in testing a biased drug is in heterologous expression systems. At this stage, it is important to use pharmacological assays that allow for an accurate assessment of bias, which in practice most often means selecting signaling assays with similar levels of amplification. If biased signaling is noted in a heterologous expression system, the candidate can then be tested in primary cells or a cell line that best models the tissue being targeted to minimize any potential system bias due to different levels of transducer expression from the heterologous expression system. Testing the ligand in human cells and/or tissue prior to direct human trials provides further validation. Before studying the compounds in model organism(s) to test safety and efficacy, it is critical to confirm that the drug binds and that bias is conserved at the receptor of a model organism. For example, a mouse receptor should be evaluated in heterologous expression systems in the same assays that a human receptor was tested. After confirming binding and biased signaling at the receptor of a model organism, the ligand can be moved ahead with more confidence. Frequently it is helpful to perform experiments with a biased candidate in parallel with balanced agonists and antagonists, to confirm that the effects of the candidate are due to bias and not to simple agonism or antagonism. The use of genetically altered animal models can provide further data about the mechanism of action. For assessing biased ligands, knocking out the receptor target can confirm that it mediates the biological effect, and removing or

altering a presumed critical transducer (such as a G protein or beta-arrestin isoform) that the candidate is biased towards can provide strong support about the specific pathway(s) involved. Caution is warranted when interpreting a phenotype from models lacking a critical transducer, even from conditional knockouts, as these transducers almost always couple to multiple receptors and signaling pathways. Some transducers have overlapping roles, such as the two isoforms of beta-arrestins, and knocking out the dominant isoform in the tissue of interest is often necessary when dual or multiple knockouts are lethal or not feasible. As in all genetic manipulations, compensation and alternative pathway selection are additional confounding factors to consider.

Table 2: Selected biased agonists under different phases of evaluation for therapeutic use.

Ligand Receptor Signaling bias Indication Development status Carvedilol Beta 1 AR Mild beta- Congestive heart Approved Beta 2 AR arrestin failure Oliceridine Mu opioid G protein Moderate to Phase III (TRV130) severe pain TRV734 Mu opioid G protein Moderate to Phase I severe pain Triazole 1.1 Kappa opioid G protein Pruritus Preclinical RB-64 Kappa opioid G protein Pain Preclinical PZM21 Mu opioid G protein Pain Preclinical UNC9994, Dopamine 2 beta-arrestin Schizophrenia Preclinical UNC9975 and mood disorders TRV250 Delta opioid G protein Migraine Preclinical

2.3 Biased Signaling at Chemokine Receptors

Until recently, it was unclear whether biased agonism was a byproduct of GPCR complexity that can only be exploited by synthetic drugs or whether it is a property that has evolved within GPCR systems as an additional layer of signaling specificity. We now appreciate that some endogenous ligands are biased agonists. One group of GPCRs where endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 chemokine receptors that bind one another with significant redundancy and promiscuity [206, 207]. For example, the chemokine receptor CXCR3-A, which plays significant roles in inflammation, vascular disease, and cancer, has four known endogenous ligands: CXCL4, CXCL9, CXCL10, and CXCL11 [208-210]. Interestingly,

CXCL11 promotes CD4+ T cell polarization into FOXP3-negative regulatory T cells via

STAT3 and STAT6 dependent pathways, while CXCL9 and CXCL10 promote CD4+ polarization into effector Th1/Th17 cells via STAT1, STAT4, and STAT5 dependent pathways [211]. Beta-arrestin- and GRK-biased chemokines have also been described at other chemokine receptors, including CXCR2, CCR1, CCR5, and CCR7 [55, 212, 213].

Similar to many chemokines, the endogenous CXCR4 ligand CXCL12 (stromal cell- derived factor 1) can exist in monomeric or dimeric forms. Monomeric CXCL12 signals through both G protein and beta-arrestin pathways at CXCR4, while dimeric CXCL12 signals through G proteins with minimal to absent beta-arrestin recruitment and signaling [214]. Demonstrating further granularity of biased signaling, different

chemokines can exhibit G protein subunit bias, as endogenous chemokines for CCR5 and CCR7 signal through overlapping but distinct G protein subtypes [215]. In addition to endogenous biased signaling, biased synthetic small molecules with affinity for chemokine receptors have also been identified [216]. Biasing chemokine receptor signaling may provide an avenue for drugging a GPCR family that has been notoriously difficult to therapeutically target.

3. CXCR3 splice variants differentially activate beta- arrestins to regulate downstream signaling pathways

This chapter describes work dissecting endogenous bias at the chemokine receptor CXCR3, and how different CXCR3 splice variants differentially couple to intracellular effectors. The following individuals made critical contributions: Priya

Alagesan, Nimit K. Desai, Thomas F. Pack, Jiao-Hui Wu, Asuka Inoue, Neil J. Freedman, and Sudarshan Rajagopal. Notably, Jiao-Hui Wu and Neil J. Freedman performed the majority of the P32 metabolic labeling experiments, Thomas Pack designed the beta- arrestin-2 biosensor, and Asuka Inoue generated beta-arrestin-1 and beta-arrestin-2 dual knockout HEK293 cells.

3.1 CXCR3 receptor splice variants

CXCR3 has two seven-transmembrane spanning splice variants, CXCR3A and

CXCR3B. CXCR3A and CXCR3B have identical intracellular sequences and only differ by the replacement of the four most distal residues on the N-terminus of CXCR3A with

51 amino acids unique to CXCR3B due to alternative splicing at the 5’ end of the second exon [217] (Figure 4). These splice variants are known to signal in response to four chemokines: CXCL4 (Platelet Factor-4; PF-4), CXCL9 (Monokine induced by IFN-

[gamma]; MIG), CXCL10 (IFN-induced protein 10; IP-10), and CXCL11 (IFN-inducible T- cell [alpha] chemoattractant; I-TAC) [218-221].

Figure 4: CXCR3A and CXCR3B splice variants. Schematic representation of CXCR3A (A) and CXCR3B (B). Light pink signifies the additional 47 amino acids present on the N-terminus of CXCR3B. There is no difference in intracellular residues between these CXCR3 splice variants. (C) Sequence alignment of CXCR3A and B.

CXCR3 was first discovered through its selective recruitment of effector T-cells

[222], and is now known to be a critical mediator of inflammation, vascular disease, and

cancer [223]. Aberrant CXCR3 signaling is implicated in inflammatory diseases, inhibition of blood vessel formation, and both tumor repression and tumorigenesis [224-

227]. Due to differential regulatory promotor elements, the endogenous ligands of

CXCR3 are spatially and temporally separated by expression pattern [209], suggesting distinct functional properties of CXCL4, CXCL9, CXCL10, and CXCL11. CXCR3A is primarily expressed on activated T-lymphocytes [228], but expression is also noted on a variety of other cell types including dendritic cells, natural killer cells, B-cells, and macrophages [229]. In contrast, CXCR3B is expressed predominantly on microvascular endothelial cells [217], as well as on T-lymphocytes, although at a lower level than

CXCR3A. A recent report demonstrated differential effects of endogenous ligands at

CXCR3B compared to CXCR3A [47], however, the role of beta-arrestin recruitment and how recruitment propagates to downstream signaling between CXCR3A and CXCR3B remains unclear. The goal of this work was to understand how the endogenous CXCR3 ligands differentially signal through G proteins and beta-arrestins, and how the different

N-termini of CXCR3A and CXCR3B influence intracellular signaling through G-proteins, beta-arrestins, and GRKs.

3.2 CXCR3B is beta-arrestin-biased compared to CXCR3A

We first examined G protein signaling at CXCR3A and CXCR3B. Prior studies identified selective affinity of CXCL4, CXCL9, CXCL10, and CXCL11 for CXCR3A and

CXCR3B through radioligand binding [217, 230]. We tested CXCR3A and CXCR3B

signaling via G[alpha]i by assessing inhibition of cyclic AMP (cAMP) production through a modified cAMP dependent firefly luciferase with the four known endogenous ligands. In agreement with previous results at CXCR3A [213], CXCL10 and CXCL11 were found to be full agonists, while CXCL9 was found to be a partial agonist, in their ability to suppress cAMP production in a highly amplified assay (Figure 5, Table 3).

Figure 5: Differential G protein activation and beta-arrestin recruitment by CXCR3 splice variants. (A) cAMP signal following transient CXCR3A expression in HEK293 cells stably expressing the cAMP activated firefly luciferase. CXCL10 and CXCL11 are full agonists in their ability to inhibit cAMP production through CXCR3A,

while CXCL4 and CXCL9 are partial agonists. (B) No G[alpha]i activity is observed after transient transfection with CXCR3B. CXCL11 recruited beta-arrestin-2-YFP to both CXCR3A-Rluc (C) and CXCR3B-Rluc (D) with higher efficacy than the other endogenous ligands. CXCL11 recruited beta-arrestin-1-YFP to CXCR3A-Rluc with a significantly greater efficacy than CXCL4, but not significantly greater than CXCL9 or CXCL10 (E). At CXCR3B, no ligands were observed to be significantly different in recruiting beta- arrestin-1-YFP (F). Best fit calculated by a 3-parameter fit, ±SEM, n≥3 biological replicates per treatment group. *, p < 0.05, significant effect of ligand by two-way ANOVA. n.s is not significant.

Slight inhibition of cAMP signal was observed with CXCL4 at high concentrations. In contrast, none of these four ligands were observed to inhibit cAMP at CXCR3B (Figure

5B, Table 3). As CXCR3B has previously been reported to couple to G[alpha]s [217], we also tested these four ligands’ ability to stimulate cAMP.

Table 3: Efficacies and potencies for chemokines at CXCR3A and CXCR3B. Emax (expressed as % of CXCL11 signal) and EC50 values of CXCL4, CXCL9, CXCL10, and CXCL11 at CXCR3A and CXCR3B calculated from a 3 parameter fit (y=Min + (Max- min)/(1+10^((LogEC50-X))). If the 3 parameter fit of ligand-receptor interaction produced a poor fit, then the data was omitted from the table. Radioligand binding data is from Lasagni et al. [217], and their data was converted to log base 10. Standard error of the mean was propagated using the equation SEM=0.434(log10 EC50 error/log10 EC50).

CXCR3A G[alpha]i Emax ± SEM Log EC50 ± SEM CXCL4 33 ± 21 -6.9 ± 1.04 CXCL9 58 ± 11 -7.1 ± 0.33 CXCL10 85 ± 9 -7.6 ± 0.21 CXCL11 100 ± 7 -7.4 ± 0.12

beta-arrestin-1 CXCL4 -16 ± 7 -7.1 ± 0.81 CXCL9 33 ± 5 -7.5 ± 0.33 CXCL10 37 ± 19 -6.5 ± 0.57 CXCL11 100 ± 12 -6.8 ± 0.17

beta-arrestin-2 CXCL4 17 ± 6 -8.6 ± 0.41 CXCL9 24 ± 5 -7.2 ± 0.42 CXCL10 37 ± 4 -7.3 ± 0.19 CXCL11 100 ± 3 -7.4 ± 0.06

Total Internalization CXCL9 69 ± 178 -6.0 ± 1.6 CXCL10 88 ± 48 -6.3 ± 0.56 CXCL11 100 ± 18 -7.0 ± 0.23

Endosome associated internalization CXCL10 57 ± 10 -7.5 ± 0.34 CXCL11 100 ± 11 -8.0 ± 0.19 beta-arrestin associated internalization CXCL11 100 ± 7 -7.8 ± 0.14

CXCR3B

beta-arrestin-2 CXCL11 100 ± 15 -7.2 ± 0.30

Total Internalization CXCL11 100 ± 24 -6.7 ± 0.56 Radioligand Binding (from [217]) Log IC50 ± SEM CXCR3A CXCL4 -6.3 ± 0.47 CXCL9 -7.5 ± 0.44 CXCL10 -9.5 ± 0.46 CXCL11 -9.4 ± 0.46

CXCR3B CXCL4 -8.1 ± 0.45 CXCL9 -6.9 ± 0.46 CXCL10 -8.2 ± 0.46 CXCL11 -7.5 ± 0.46

No signal was observed with CXCL4, CXCL9, CXCL10, or CXCL11 while a positive control of 100nM isoproterenol demonstrated a ~15 fold increase in signal from baseline. We then used BRET to test beta-arrestin-1 and beta-arrestin-2 recruitment to

CXCR3A and CXCR3B following CXCL4, CXCL9, CXCL10, and CXCL11 stimulation

(Figure 5C-F). At CXCR3A, we found that all the ligands recruited beta-arrestin-2 with rank order of efficacy of CXCL11 > CXCL10 > CXCL9 > CXCL4 (Figure 5C). A bias plot of relative intrinsic activity [200] displayed divergent efficacy of CXCL11 towards the beta-arrestin-2 pathway relative to the other three endogenous CXCR3 ligands (Figure

6). At CXCR3B, only CXCL11 recruited beta-arrestin-2, with no significant recruitment noted for the other endogenous ligands (Figure 5D). The association of beta-arrestin-2 with CXCR3A was of longer duration than with CXCR3B (Figure 7). CXCL4 displayed inverse agonist characteristics for beta-arrestin-2 recruitment to CXCR3B, with significantly less signal than CXCL9, which displayed no significant activity. CXCL11 recruited beta-arrestin-1 to a significantly greater efficacy to CXCR3A than other ligands

(Figure 5E). No statistically significant interaction of ligand and concentration was observed for beta-arrestin-1 recruitment to CXCR3B (Figure 5F). These findings are

consistent with those of Berchiche et al. who found that CXCR3B acted as a beta- arrestin-biased receptor relative to CXCR3A [47].

Figure 6: Bias plot at CXCR3A. Bias plot of CXCL4, CXCL9, CXCL10, and CXCL11 for G[alpha]i (x-axis) and beta-arrestin-2 (y-axis). These plots allow for the visualization of relative bias without designating a single ‘reference’ agonist, ideal for a system with multiple endogenous ligands. Increased efficacy of CXCL11 towards beta- arrestin-2 relative to CXCL10, CXCL9, and CXCL4 is apparent. Given the absence of observed G[alpha]i signal at CXCR3B, we are unable to provide a bias plot for that isoform.

Figure 7: Differential duration of beta-arrestin-receptor association. (A) Schematic of BRET assay used to quantify beta-arrestin recruitment. The donor Rluc was added to the C-terminus of CXCR3A and CXCR3B. The acceptor YFP was added to the C-terminus of beta-arrestin-1 and beta-arrestin-2. (B) Association of beta-arrestin-2 at either CXCR3A or CXCR3B at different times after CXCL11 stimulation as quantified by Net BRET Ratio. No significant decay in association was observed at CXCR3A- beta- arrestin-2 at 15 minutes post stimulation, while a significant decay was observed at CXCR3B compared to both 5 minute and 10 minute CXCL11 stimulation durations. *, p < 0.05 by one-way ANOVA with Tukey’s post-hoc comparison to both 5 and 10 minutes.

3.3 Both CXCR3A and CXCR3B undergo agonist-dependent phosphorylation

We next focused on assessing whether the mechanisms underlying beta-arrestin recruitment to CXCR3A and CXCR3B differed from each other. Phosphorylation of intracellular residues is necessary to recruit beta-arrestins. Given the differential isoform trafficking, we examined agonist-induced phosphorylation of both CXCR3A and

CXCR3B by 32P metabolic labeling. CXCL11 treatment resulted in phosphorylation of

both receptor isoforms to comparable levels (Figure 8), consistent with the observation that beta-arrestin-2 is recruited to both receptors. Based on the CXCR3 immunoblot, much of the expressed receptor is not glycosylated; the phosphorylated receptor corresponds to the glycosylated form of the receptor that would be expressed on the plasma membrane [231]. While a slight difference in mobility of CXCR3B is present in the unglycosylated form, which is likely related to the small predicted shift in molecular weight, there was no significant difference in the mobility of the glycosylated splice variants.

3.4 Activation of CXCR3A and B generate distinct patterns of beta-arrestin recruitment and conformation

We then examined CXCL11-induced beta-arrestin-GFP trafficking in cells transfected with CXCR3A or CXCR3B by confocal microscopy (Figure 9). At CXCR3A, beta-arrestin-2-GFP was strongly recruited to the plasma membrane immediately following treatment with 100 nM CXCL11, with beta-arrestin-2-GFP complexes forming in the cytosol in a ‘class B’ recruitment pattern 30-40 minutes post ligand treatment. In contrast, CXCL11 treatment of CXCR3B-transfected cells only induced weak beta- arrestin-2-GFP recruitment to the plasma membrane in a ‘class A’ pattern [165], with maximal beta-arrestin-2-GFP complex formation at the cell membrane observed 20-30 minutes post stimulation. No appreciable beta-arrestin-2-GFP complexes in CXCR3B- transfected cells were observed in the cytosol at 40 minutes or longer.

Figure 8: CXCR3A and CXCR3B demonstrate equivalent agonist-induced phosphorylation. HEK293 cells were transfected with plasmids encoding either no protein (“Vector”) or N-terminal FLAG-tagged constructs of CXCR3A or CXCR3B. After serum starvation and metabolic labeling with 32Pi, cells were exposed to serum-free medium lacking (“-”) or containing (“+”) the CXCR3 agonist CXCL11 (100 nM) for 5 min (37 ºC), and then solubilized. CXCR3 isoforms were immunoprecipitated (IP) via their N-terminal FLAG epitope, and immune complexes were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and then subjected sequentially to autoradiography (“32P”) and immunoblotting (IB) for CXCR3, as indicated. Shown are an autoradiogram and immunoblot from a single experiment, representative of 3 performed. The arrow indicates the cell-surface, mature-glycosylated isoform of CXCR3 (which has slower electrophoretic mobility than the cotranslationally glycosylated or

immature-glycosylated bands). From the 32P signal for each CXCR3 band we subtracted the (nonspecific) signal in the cognate location of the lane from untransfected cells; the resulting specific 32P CXCR3 signal was normalized to the cognate CXCR3 immunoblot band signal (from which nonspecific pixels in the cognate untransfected cell lane had been subtracted). In each experiment, this ratio of 32P/CXCR3 was normalized to that obtained for CXCR3B immunoprecipitated from unstimulated cells, to obtain “fold/control”, plotted as individual values and corresponding means ± SEM. Compared with unstimulated cells: *, p < 0.05 indicates a significant effect of agonist by two-way ANOVA, with no significant effect of receptor isoform. n=3 biological replicates per treatment group.

Figure 9: CXCR3A and CXCR3B induce distinct beta-arrestin-2 trafficking patterns. Confocal microscopy of HEK 293 cells transiently transfected with unlabeled CXCR3A or CXCR3B and beta-arrestin-2-GFP pre- (left panels) and post- (right 3 panels) CXCL11 treatment (100 nM). Three different cells are shown post treatment for each splice variant.

To assess whether CXCR3A and CXCR3B generate distinct beta-arrestin conformations, we utilized a beta-arrestin-2 biosensor consisting of a nanoluc (Promega) donor linked to the N-terminus and a YFP acceptor on the C-terminus of beta-arrestin-2, which produces a higher intensity signal than a previously-designed beta-arrestin

biosensor [67]. A significant change in the magnitude of the BRET ratio could be due to a change in distance and/or dipole orientation between the donor and acceptor or a change in avidity in the beta-arrestin-receptor interaction.

Figure 10: Limiting beta-arrestin-2 biosensor avidity. Biosensor titration response cotransfected with CXCR3A to determine optimal biosensor transfection. CXCR3A and biosensor were transiently cotransfected into 293T cells and stimulated with CXCL11 (250 nM) for 15 minutes. Low concentration (10 ng) transfections produced poor signal to noise, while higher amounts of biosensor expression vector resulted in reduced signal following ligand stimulation. *, p < 0.05 by one-way ANOVA with Dunnett’s post-hoc comparison to 50 ng transfections.

We performed a dose-receptor titration of the biosensor to find the optimal biosensor concentration that attempts to limit avidity effects in our assay by utilizing the lowest concentration of biosensor that produced a reliable signal and reducing excess biosensor remaining in the cellular pool (Figure 10). Therefore, the changes seen in the net BRET ratio primarily imply a conformational change of beta-arrestin-2. We transiently transfected cells with this biosensor (Figure 11A) as well as untagged

CXCR3A or CXCR3B and subsequently stimulated with CXCL4, CXCL9, CXCL10, or

CXCL11. We detected a significant change in BRET signal in the beta-arrestin-2 biosensor after stimulation of CXCR3A with CXCL11 (Figure 11B), but not after stimulation of CXCR3B (Figure 11C). This suggests that stimulation of CXCR3A with

CXCL11 leads to a distinct change in beta-arrestin conformation.

Figure 11: CXCR3A, but not CXCR3B, stimulation with CXCL11 causes a change in beta-arrestin-2 conformation. (A) Schematic of beta-arrestin-2 biosensor for probing beta-arrestin-2 conformational changes following transfection with the biosensor and untagged CXCR3A or CXCR3B. HEK293 cells were stimulated for 15 minutes with the indicated ligand (250 nM) prior to BRET measurements. (B) CXCL11 stimulation of CXCR3A led to a significant conformational change in beta-arrestin signified by a change in magnitude of the net BRET ratio compared to all ligands, while CXCL10 stimulation caused a significantly different signal from CXCL4. (C) Stimulation of CXCR3B resulted in no appreciable change in signal across all comparisons. *, p < 0.05 by one-way ANOVA with Tukey’s post hoc comparison between all treatment groups. n≥3 biological replicates per condition.

3.5 CXCR3 splice variants differentially internalize

Receptor internalization is one of the most well-appreciated functions of beta- arrestins. Because beta-arrestin recruitment, as assessed by BRET, does not necessarily correlate with function, we next investigated if CXCL11 stimulation resulted in a functional difference compared to the other three endogenous ligands. The earlier study by Berchiche et al. demonstrated that both CXCR3A and B undergo similar agonist- induced internalization, although the kinetics for internalization depended on the specific agonist. However, there are distinct mechanisms for GPCR internalization, which can be dependent or independent of beta-arrestins, which can act as clathrin adapters [59]. Therefore, we quantified receptor internalization in three separate assays:

1) a bystander BRET-based membrane dissociation assay, in which receptor-Rluc internalization results in a decrease in the net BRET ratio from myr-palm labeled mVenus (Figure 12A, middle panel); 2) a bystander BRET-based early endosome association assay, in which receptor-Rluc association with 2x-FYVE labeled mVenus localized to causes an increase in the net BRET ratio (Figure 12A, right panel); and 3) a beta-arrestin-complex mediated internalization using an assay in U2OS cells permanently expressing split beta-galactosidase fragments on beta-arrestin-2 and endosomes (DiscoveRx). In this last assay, when beta-arrestin-2 and endosomes are in close proximity for a sustained duration, complementation of beta-galactosidase fragments in the presence of a substrate produces a chemiluminescent signal. Signal is

produced in ‘Class B’ stable endosome beta-arrestin-2 interactions, however, limited or no signal is produced in ‘Class A’ transient beta-arrestin-2 -endosome interactions.

Figure 12: Receptor internalization. (A) Confocal images of transiently transfected untagged mVenus (left panel), myr-palm-mVenus (center panel) localized to 61

the plasma membrane, and 2x-FYVE-mVenus (left panel) localized in an endosomal distribution. Internalization efficacy measured by BRET of myr-palm-mVenus transiently transfected cells with (B) CXCR3A-Rluc or (C) CXCR3B-Rluc CXCL11 and treated with the indicated ligand for one hour as a measure of total receptor internalization. 2x-FYVE-mVenus transiently transfected cells (D) CXCR3A-Rluc or (E) CXCR3B-Rluc and treated with the indicated ligand for one hour as a measure of receptor-endosome association. Treatment CXCL11 resulted in greater beta-arrestin- mediated internalization efficacy compared to all other ligands in cells transiently transfected with CXCR3A (F), but not CXCR3B (G). Aside from panel G which lacked appreciable signal with any ligand, data are normalized to CXCL11 (1 uM) stimulation and expressed as a percentage of maximal signal. *, p < 0.05, significant effect of ligand by two-way ANOVA. Scale bar is 10 uM. n≥3 biological replicates per condition.

At CXCR3A, total internalization, early endosome association, and beta-arrestin associated internalization at CXCR3A mirrored rank order of efficacy of beta-arrestin-2 recruitment, with the order of CXCL11 > CXCL10 > CXCL9 > CXCL4. CXCL4 stimulation resulted in negligible internalization and displaying weak inverse agonist activity in the early endosome association assay (Figure 12B, D, F). At CXCR3B, only CXCL11 induced statistically significant total internalization and early endosome association (Figure 12C,

E). No CXCR3B-mediated chemiluminescent signal in the beta-arrestin-mediated internalization assay was observed (Figure 12G), consistent with the ‘Class A’ pattern of beta-arrestin recruitment noted on confocal imaging (Figure 9). Thus, although both receptors undergo agonist-induced internalization, they appear to do so using different underlying mechanisms.

3.6 C-terminus truncation and GRK knockdown reduce beta- arrestin-2 recruitment

Given our findings in the GRK BRET experiments, we hypothesized that inhibition of GRKs would attenuate beta-arrestin-2 recruitment at CXCR3A. We first generated C-terminal truncation mutants of CXCR3A and CXCR3B that lack putative C- terminal phosphorylation sites (Figure 13A). The mutants’ truncation location is at the identical C-terminal residue given the extended N-terminus of CXCR3B (344th residue on CXCR3A, 391st residue on CXCR3B). Truncation resulted in ~50% reduction, but not elimination, of beta-arrestin-2 recruitment efficacy to both CXCR3A and CXCR3B as quantified by BRET (Figure 13B), similar to other GPCRs such as the Angiotensin Type

1A receptor [232], but unlike other GPCRs, such as CCR5 and the Apelin receptor, where beta-arrestin-2 recruitment is not observed after removal of the C-terminus [40, 233].

Given this data, CXCR3 C-terminal phosphorylation-dependent beta-arrestin-2 recruitment would not be expected to completely eliminate beta-arrestin-2 recruitment to the receptor. Indeed, siRNA knockdown of either GRK2/3 or GRK5/6 decreased, but did not eliminate, CXCR3A – beta-arrestin-2 association following CXCL11 stimulation as measured by BRET (Figure 13C). Interestingly, GRK2/3, but not GRK5/6, siRNA knockdown prominently attenuated CXCR3B-beta-arrestin-2 association (Figure 13D).

This suggests, although both receptor splice variants undergo agonist-dependent phosphorylation, that different GRKs are playing distinctive roles at each receptor.

Figure 13: GRK5/6 knockdown differentially attenuates beta-arrestin2 recruitment. (A) Cartoon of CXCR3 truncation C-terminal mutants. (B) Truncation of either CXCR3A or CXCR3B reduced beta-arrestin-2 recruitment as measured by BRET in response to CXCL11 (500 nM). (C) siRNA knockdown of either GRK2/3 or GRK5/6 reduced CXCL11 (500 nM) induced beta-arrestin-2 recruitment to CXCR3A, however, (D) only siRNA knockdown of GRK2/3, but not GRK5/6, reduced beta-arrestin-2 recruitment to CXCR3B. For panel B, *, p < 0.05 by unpaired student’s t-test between respective WT and truncation mutant. For panels C and D, *, p < 0.05 by one-way ANOVA with Tukey’s post hoc comparison between all treatment groups. n≥3 biological replicates per condition.

3.7 CXCL11 activates ERK 1/2 at both CXCR3A and CXCR3B

G proteins and beta-arrestins can activate similar intracellular pathways, but with different spatial and temporal patterns that result in distinct cellular responses [4,

59]. One well-characterized example is activation and phosphorylation (pERK) of the

ERK 1/2 MAP kinases (MAPKs) [30]. For many receptors, such as the Angiotensin Type

IA and Parathyroid hormone receptors, G proteins, beta-arrestin-1, and/or beta-arrestin-

2 significantly contribute to early phase pERK [135, 234, 235], while at others, G protein- mediated pERK includes both a ‘fast’ and ‘slow’ phase [167]. Thus, kinetics alone cannot distinguish between G protein- and beta-arrestin-dependent pERK, and the contributions of these different pathways to the pERK response requires more detailed characterization. We observe that agonist-induced activation of CXCR3A and CXCR3B results in different intensities of activation with similar kinetics at early time points, as the pattern of pERK activation at 5 minutes by the four endogenous ligands was not different between HEK293 cells transiently transfected with CXCR3A or CXCR3B

(Figure 14A and C). Given that differential beta-arrestin signaling often occurs at time points beyond 15 minutes, we evaluated signaling up to one hour. Interestingly, a differential pattern of signaling emerged between splice variants at the one-hour time point, as CXCR3A transfected cells showed greater ERK activation at 60 minutes following stimulation with CXCL11, while CXCR3B transfected cells did not (Figure 14B,

D).

Figure 14: Divergent ERK activation kinetics between CXCR3 splice variants. HEK293 cells were transfected with either CXCR3A or CXCR3B and stimulated for either 5 min (A) or 60 min (B) with the indicated ligand (100 nM). The 60 minute, but not 5 minute, phospho-ERK 1/2 CXCL11 signal in cells transfected with CXCR3A was significantly greater than cells transfected with CXCR3B. (C, D) Quantification of phospho-ERK/ ERK signal normalized to CXCR3A vehicle treatment. Data were

normalized to vehicle treatment of each respective isoform. In panels C and D, *, p < 0.05 indicates a significant effect of transfected isoform at 60 minutes by two-way ANOVA. #, p <0.05; by one-way ANOVA and Tukey post hoc comparisons within all CXCR3A treatment groups, with ‘*’ indicating a significant difference between of CXCR3A CXCL11 stimulated cells compared to vehicle and CXCL4 at both 5 and 60 minutes; ‡, p < 0.05, by one-way ANOVA and Tukey post hoc comparisons between all CXCR3B treatment groups, with ‘‡’ indicating a significant difference between CXCR3B CXCL11 stimulated cells compared to all treatment groups at 5 minutes. No significant differences between ligands were observed in CXCR3B transfected cells at 60 minutes. ±SEM, n≥3 biological replicates per condition. Western blots shown are representative of at least 3 separate experiments.

3.8 CRISPR/Cas9 knockout of beta-arrestins differentially affects CXCR3 splice variants

Given the differential pattern of observed ERK activity, we tested the effects of beta-arrestin knockout on ERK activation by CXCR3 splice variants. To perform this, we used a different HEK 293 cell line in which both beta-arrestin-1 and beta-arrestin-2 (Arrb

1/2 knockout (KO)) were removed by CRISPR/Cas9 genome editing. Beta-arrestin-1 and beta-arrestin-2 knockout was confirmed by immunoblot with polyclonal anti-beta- arrestin antibodies recognizing either the C-terminus of beta-arrestin-1 (A1-CT) or the C- terminus of beta-arrestin-2 (A2-CT) (Figure 15).

Figure 15: Confirmation of beta-arrestin knockout. Beta-arrestin expression in WT and ∆ARRB1/2 HEK293 cells, demonstrating lack of beta-arrestin-1 and beta- arrestin-2 in the CRISPR ARRB1/2 KO cells.

Phospho-ERK signal in these Arrb 1/2 KO cells was compared to WT cells following 100nM CXCL11 stimulation. Both WT and Arrb 1/2 KO cells from this HEK

293 line differed in morphology and growth rate compared to HEK 293 cells used in other experiments. The basal pERK activity of the Arrb 1/2 KO vehicle control group of both CXCR3A and CXCR3B transfected cells was greater than in identically treated WT cells (Figure 16A, B). In both CXCR3A transfected WT and Arrb 1/2 KO cells, CXCL11 at

5 minutes caused a significant increase in pERK signal (Figure 16A, C). In contrast, at

CXCR3B, WT cells displayed a significant increase in pERK signal compared to vehicle while Arrb 1/2 KO cells did not (Figure 16B, D). The residual ERK phosphorylation by

CXCR3B in Arrb 1/2 KO cells may represent very low residual G[alpha]i signaling not detected in our second messenger assays or coupling to alternative G proteins. To further probe the differential pERK signal observed between WT and Arrb 1/2 KO cells

at 5 minutes expressing either CXCR3A or CXCR3B, we rescued beta-arrestin-1 and beta-arrestin-2 through transient overexpression in the Arrb 1/2 KO cells and compared pERK signal at 5 minutes to empty vector transfection controls.

Figure 16: Beta-arrestin knockout differentially alters pERK signaling between CXCR3 splice variants. WT or ∆ARRB1/2 cells transfected with CXCR3A (A), or CXCR3B (B), and stimulated with CXCL11 (100nM). A significant increase in phospho- ERK 1/2 signal was observed in both WT and ∆ARRB1/2 cells transfected with CXCR3A. However, only WT, but not ∆ARRB1/2, cells transfected with CXCR3B displayed a

significant increase in phospho-ERK 1/2 signal. CXCR3A (C) and CXCR3B (D) phospo- ERK signal was quantified in WT and ∆ARRB1/2 cells (please note the change in scale of the y-axis between panels C and D). In panels C and D, &, p < 0.05 indicates a significant effect of cell line by two-way ANOVA in cells transfected with CXCR3B; *, p < 0.05 indicates a significant difference of the 5 min time point by two-way ANOVA followed by Bonferroni post hoc comparison (corrected for all time points) in cells transfected with CXCR3B; #, p < 0.05 by one-way ANOVA and Dunnett’s post hoc comparison of CXCL11 stimulation to the respective isoform vehicle control at the indicated time point. ±SEM, n≥3 biological replicates per condition. Western blots shown are representative of at least 3 separate experiments.

Similar to the trend observed between WT and Arrb 1/2 KO cells, 5-minute pERK signal in CXCR3A transfected cells was significantly decreased by beta-arrestin overexpression rescue relative to vehicle (Figure 17A, D). Conversely, the pERK signal was significantly potentiated in CXCR3B transfected cells relative to vehicle (Figure 17B,

D), with a significant difference also noted between isoforms (Figure 17D).

Figure 17: Beta-arrestin overexpression differentially regulates pERK signaling between CXCR3 splice variants at 5 minutes. Either beta-arrestin-1 and beta-arrestin-2 or empty vector were transfected in ARRB1/2 KO cells expressing either (A) CXCR3A or (B) CXCR3B as a rescue overexpression experiment and stimulated for 5 minutes with either vehicle or CXCL11 (100 nM). (C) Rescue of beta-arrestin was confirmed (nonspecific band in both lanes noted with an arrow). (D) Immunoblots were quantified by calculating phospho-ERK / total ERK signal. Beta-arrestin rescue resulted in decreased phospho-ERK signal in CXCR3A expressing cells, in contrast to an increased signal in CXCR3B expressing cells, relative to respective vehicle treatments. In panel D, #, p < 0.05 indicates a significant difference in pERK signal between pcDNA empty vector and beta- arrestin rescue within isoform treatment by two-way ANOVA followed by Bonferroni post hoc comparison, while *, p < 0.05 indicates a significant difference in pERK signal between receptor isoforms treated with CXCL11 by two-way ANOVA followed by Bonferroni post hoc comparison. ±SEM, n≥3 biological replicates per condition. Western blots shown are representative of at least 3 separate experiments. IB is immunoblot.

3.9 Differential transcriptional regulation by CXCR3 splice variants

When assessing bias, testing activity distal to direct receptor transducers is helpful in modeling physiological relevance. For example, if biased signaling is observed at proximal effectors such as G proteins and beta-arrestins, but not distal transducers such as regulators of transcription, the significance of the observed bias is less clear. We further probed distal signaling by evaluating transcriptional activation through serum response reporter assays. Serum response element SRE is known to respond to the ternary complex (TCF) dependent ERK/MAPK activity, while serum response factor- response element (SRF) is a mutant form of SRE lacking the TCF binding domain that also responds to SRF dependent and TCF independent signals, such as RhoA [236, 237].

Either SRE or SRF reporters were transiently cotransfected with either CXCR3A or

CXCR3B. Transcriptional activity was assessed following incubation with the four

CXCR3 endogenous ligands. Stimulation with CXCL11 resulted in robust signal from both SRE and SRF-RE reporters co-transfected with CXCR3A (Figure 18A, C), but not when co-transfected with CXCR3B (Figure 18B, D). Both SRE and SRF signals were attenuated, but not eliminated, by mitogen activated protein kinase kinase (MEK) inhibition prior to CXCL11 stimulation (Figure 18E, F). This is consistent with ERK activation at later time points resulting in some transcriptional regulation of SRE and

SRF.

Figure 18: CXCL11 robustly activates serum response element (SRE) and serum response factor (SRF) response element signaling at CXCR3A, but not CXCR3B. HEK293T cells were transiently transfected with either the SRE or SRF reporter and either CXCR3A or CXCR3B. Prior to acquiring luminescence signal, cells were incubated for 5 hours with ligand (1 uM). CXCL11 incubation caused a significant increase in luminescence signal in both SRE (A) and SRF (C) transfected cells. In cells transfected with CXCR3B, none of the endogenous ligands tested resulted in significant SRE (B) or SRF (D) signal, although cells still responded to the positive FBS control. In panels E-F, cells were pretreated either with vehicle or with MEK inhibitor PD98059 (20 uM). The CXCL11 induced SRE and SRF signals were sensitive to MEK inhibition (E, F). *, p < 0.05; for panels A-D, a one-way ANOVA and Tukey post hoc comparisons of treatment groups. The positive control of 10% FBS is included for reference, but not included in statistical analyses. For panels E and F, a one-way ANOVA followed by a directed Bonferroni post hoc comparison of Veh+CXCL11 to PD98059+CXCL11 was conducted. n≥3 biological replicates per condition.

3.10 Discussion of CXCR3 splice variants

In this research, we found that CXCR3 receptor splice variants displayed biased signaling that was associated with distinct beta-arrestin conformations and signaling to downstream pathways, including ERK and SRE/SRF. Unlike CXCR3A, which demonstrated robust signaling through G[alpha]i, CXCR3B did not display any appreciable G[alpha]i protein signaling in second messenger assays. In rank order of efficacy, CXCL11, CXCL10, and CXCL9, but not CXCL4, stimulation caused internalization of CXCR3A in the same order previously described in human T lymphocytes and HEK cells [47, 238]. This internalization rank order was identical to the rank order of efficacy in beta-arrestin-2 recruitment to CXCR3A as measured by BRET.

Interestingly, only CXCL11, and not the other CXCR3 ligands recruited beta-arrestin-2 to

CXCR3B and resulted in measureable receptor internalization. The CXCR3B-beta- arrestin interaction was confined to the plasma membrane in a ‘class A’ pattern compared to the CXCR3A-beta-arrestin interaction displaying an endocytic ‘Class B’ pattern [239]. Notably, a biosensor demonstrated distinct beta-arrestin conformations associated with these two patterns of recruitment, with distinct beta-arrestin conformations recently to correlate with distinct beta-arrestin functions at the beta-2AR

[240]. Functional activity of CXCR3B was only observed when stimulated by a ligand that caused beta-arrestin-2 recruitment, CXCL11. In further support of CXCR3B acting as a beta-arrestin-biased receptor, beta-arrestin-1 and-2 overexpression in Arrb 1/2 KO cells

increased signaling by CXCR3B while decreasing signaling by CXCR3A. Taken in its entirety, these findings suggest that CXCR3B acts as a beta-arrestin-biased receptor relative to CXCR3A, albeit with a less stable interaction with beta-arrestin compared to

CXCR3A (summary in Figure 19). This bias may be encoded at least in part by differential GRK recruitment, as siRNA knockdown of GRK5/6 attenuated beta-arrestin recruitment to CXCR3A, but not to CXCR3B.

Phosphorylation of MAP kinases is a well-studied signaling response regulated by G proteins and beta-arrestins downstream of GPCRs [4, 59]. Although pERK signal was diminished in CXCR3B-transfected Arrb 1/2 KO cells, the signal 5 minutes after stimulation was still higher than vehicle. This suggests that while CXCR3B is biased towards beta-arrestin signaling compared to CXCR3A, CXCR3B signaling does not absolutely require beta-arrestins. CXCR3B may couple to a different G protein, an unidentified signaling effector, or both. Further investigation will be needed to understand how N-terminal splice variants alter intracellular effector coupling. CXCL11 stimulation resulted in phosphorylation of both receptor isoforms to comparable levels, suggesting that the differential signaling by CXCR3A and B is due to the phosphorylation of different residues in the C-terminus and/or different conformational changes induced in the receptors.

In our studies, we focused on receptor coupling to two GRK receptor families,

GRK2/3 and GRK5/6, because GRK2- and GRK6-deficient lymphocytes display defective

trafficking, and because the expression of both CXCR3A and CXCR3B is high in lymphocytes [217, 241, 242]. Phosphorylation by different kinases of serines and/or threonines in GPCR intracellular loops and/or the C-terminus is often necessary for recruitment of beta-arrestins to the receptor [243, 244]. Our siRNA knockdown studies demonstrate a role for both GRK2/3 and GRK5/6 in CXCL11 mediated beta-arrestin-2 recruitment to CXCR3A, but a role only for GRK2/3 in beta-arrestin-2 recruitment to

CXCR3B. At other GPCRs, such as the angiotensin AT1R [60], the beta-2 adrenergic receptor [62], and CCR7 [55], different GRKs have been demonstrated to perform signaling or desensitization functions. The activity of these different kinases is thought to result in a phosphorylation ‘bar code’ of the third intracellular loop and C-terminal tail of other GPCRs that regulates beta-arrestin activity [62, 245]. Indeed, the C-terminus and third intracellular loop of CXCR3 is known to be necessary for chemotaxis, calcium flux, and internalization of CXCR3A, processes that are known to be mediated by beta- arrestins [246]. Once activated, beta-arrestin is thought to form a core scaffolding complex, which activates ERK1/2 by phosphorylation [17]. The findings here support a model for a CXCR3 signaling ‘barcode’, in which post-translational modifications in the receptor C-terminus regulate the affinity of beta-arrestin recruitment and the pattern of

GPCR intracellular trafficking [165, 247]. However, future studies that clearly link the phosphorylation of specific sites in the receptor by distinct kinases will be needed to test this hypothesis at CXCR3.

It is somewhat surprising that an N-terminal CKR modification has such an impact on receptor coupling to G proteins, GRKs and beta-arrestins. This is likely due to the mode of chemokines binding to their CKRs, which is primarily mediated by distinct chemokine recognition sites (CRS) [248]. Our current understanding of chemokine:CKR binding, which is informed both by earlier biophysical studies [249] and now crystal structures [248, 250, 251], is that the N-terminus of the chemokine inserts itself in the transmembrane regions of the CKR and interacts through an interface termed CRS2. The

C-terminal domain of the chemokine interacts with the extracellular N-terminus of the receptor through the CRS1 site. Recent crystal structures have demonstrated that the extracellular loops of the CKR interact with the chemokine through a CRS1.5 site. Post- translational sulfation of two sites on the N-terminus of CXCR3A (Tyr 27 and Tyr 29) is necessary for receptor function following ligand stimulation [252]. The extended N- terminus of CXCR3B adds two additional sulfation sites (Tyr 6 and Tyr 40). We speculate that this extended N-terminus of CXCR3B binds to its ligands through the

CRS1 site or an alternate surface on the chemokine, thereby allosterically coupling with

CRS2 site to generate distinct receptor:ligand:transducer conformations.

3.11 Summary of differences between CXCR3 splice variants

Figure 19: Summary of observed CXCR3 isoform signaling. Both CXCR3A and CXCR3B recruited beta-arrestin-2, became phosphorylated, and internalized in response to CXCL11. In contrast, only CXCR3A was observed to signal through Gai, as CXCR3B transfected cells did not produce appreciable signal in either Gai or Gas assays. In further signaling divergence, CXCR3A and CXCR3B show distinct patterns of downstream signaling, with CXCR3A observed to display a stable ‘class B’ interaction with beta-arrestin-2 in contrast to CXCR3B which displayed a transient ‘class A’ interaction in confocal recruitment assays. GRK2/3 siRNA knockdown attenuated beta- arrestin-2 recruitment to both receptor isoforms, however, only GRK5/6 knockdown was observed attenuate beta-arrestin-2 recruitment to CXCR3A. Only CXCR3A was observed to show significant late phase (one hour) phospho-ERK activity, and overexpression rescue of beta-arrestin attenuated early phase phospho-ERK activity mediated by CXCR3A while enhanced phospho-ERK activity mediated by CXCR3B. SRE and SRF transcriptional reporter activity was only observed downstream of CXCR3A.

CXCR3 splice variants CXCR3A and CXCR3B, differing by an extended N- terminus at CXCR3B, demonstrate significantly biased signaling responses. Unlike signaling observed at CXCR3A, beta-arrestin-2, but not G[alpha]i protein signaling, was detected at CXCR3B. Different patterns of receptor-beta-arrestin-2 interaction were also observed between splice variants, with CXCR3A displaying a more stable interaction with arrestin compared to CXCR3B. siRNA knockdown of GRK5/6 attenuated beta- arrestin-2 recruitment at CXCR3A, but not CXCR3B. In addition, the splice variants had distinct internalization profiles that correlated with differences in late-phase ERK activation and transcriptional activity. At this time, it is unclear as to the mechanisms underlying the phosphorylation ‘barcode’ of the CXCR3 C-terminus and which phosphorylation sites are critical for activation of G protein- and beta-arrestin-mediated pathways downstream of the receptor. Appreciating the signaling differences between these splice variants may provide clarification to conflicting reports of CXCR3 function and offer a compelling example of how the GPCR extracellular residues can dramatically change intracellular pathway activation.

4. CXCR3 in Physiology and Disease

4.1 Introduction to physiological processes regulated by CXCR3

This chapter focuses on CXCR3-A, the dominant CXCR3 isoform that is expressed primarily on activated effector memory T cells and plays an important role in

diseases including atherosclerosis, cancer, and inflammatory disorders. Unless otherwise noted in this chapter, CXCR3 refers to ‘CXCR3-A.’

Activation of CXCR3 by chemokines causes migration of activated T cells in a concentration-dependent manner. Increased tissue concentrations of activated T cells initiate inflammatory responses, and the ability to modulate T cell chemotaxis would likely be therapeutically useful in many disease processes. Despite the importance of the

> 20 CKRs in a wide variety of disease states, there are currently only three FDA- approved drugs that target CKR family members [209, 253, 254]. The difficulty in successfully targeting CKRs was originally thought to be due to ‘redundancy’ across the multiple chemokine ligands and CKRs that bind one another [255]. However, this presumed redundancy appears to be more granular than initially appreciated. Like most other chemokine receptors, CXCR3 signals through G[alpha]i G proteins and beta- arrestins. As discussed in detail within the introduction to this dissertation, GPCR signaling deviates at critical junctions, including G protein and beta-arrestins, which signal through distinct intracellular signaling pathways. For example, beta-arrestins promote interactions with kinases distinct from isolated GPCR–G protein interactions to induce downstream signaling [256].

My work, and that of others, demonstrates that many chemokines that bind to the same CKR can selectively activate such distinct signaling pathways downstream of the receptor and act as biased agonists (please refer to Chapter 3 for more detail) [55,

213, 214]. Biased ligands at other GPCRs, such as the mu-opioid receptor [185, 257], the kappa opioid receptor [258], and type 1 angiotensin II receptor (AT1R) [259], have shown promise of improving efficacy while reducing side effects through differential activation of G protein and beta-arrestin signaling [260]. Both animal and human studies suggest that G protein signaling at the mu-opioid receptor primarily mediates analgesic efficacy while beta-arrestin signaling mediates many adverse effects such as respiratory depression, constipation, tolerance, and dependence [185, 257]. Furthermore, relative degrees of G protein and beta-arrestin-bias can predict safer mu-opioid analgesics [261].

At the AT1R, biased and balanced AT1R agonists have been shown to have distinct physiologic responses: G[alpha]q signaling has been shown to mediate vasoconstriction and cardiac hypertrophy, while beta-arrestin-signaling activates anti-apoptotic signals and promotes calcium sensitization [259]. At CKRs, both pertussis toxin (PTX)-sensitive G protein signaling and beta-arrestin signaling are known to contribute to chemotaxis [212,

241, 262-265]. However, chemokines with distinct G protein and beta-arrestin-biased signaling often induce chemotaxis to similar degrees [55]. The relative contribution of beta-arrestin or G protein signaling to chemotaxis and inflammation is unclear, and it is experimentally challenging to discern the physiological relevance of biased signaling with peptide agonists in many assays due to the high molecular weight and short half- life of chemokines relative to small molecules. Indeed, it is unknown if endogenous or synthetic CKR ligands that preferentially target G protein or beta-arrestin pathways

would result in different physiological outcomes in models of disease and inflammation.

If such differences in selective pathway activation result in distinct physiological outcomes, then biased agonism could be used to develop new insights into chemokine biology that could be harnessed to increase therapeutic utility of drugs targeting CKRs while reducing on-target side effects.

4.2 Allergic contact dermatitis: a disease model for CXCR3 signaling

How can we best assess the physiological outcomes of CXCR3 biased signaling?

One disease where CXCR3 signaling appears to be critical is allergic contact dermatitis.

Allergic contact dermatitis (ACD) is a common skin disease that results in significant cost and morbidity. Despite its high prevalence, therapeutic options are limited. Allergic contact dermatitis is regulated primarily by T cells within the adaptive immune system.

The chemokine receptor system, consisting of chemokine peptides and chemokine G protein coupled receptors, is a critical regulator of this disease. Specific chemokine signaling pathways are selectively upregulated in allergic contact dermatitis, most prominently CXCR3 and its endogenous chemokines CXCL9, CXCL10, and CXCL11.

This section will summarize the pathophysiology of allergic contact dermatitis and describe why I believe it is a valid model to dissect the relative contributions of G protein and beta-arrestin signaling pathways in a physiologically-relevant context.

4.2.1 Summary of allergic contact dermatitis

Allergic contact dermatitis is mediated by many cell types within the immune system. Major cell types that contribute to the development of inflammation in allergic contact dermatitis include dendritic cells, TH1, TH17, and regulatory T cells (Tregs) [266-

269]. Effector and memory T lymphocytes are the key adaptive immune mediators of allergic contact dermatitis [270]. This differs from irritant contact dermatitis (ICD), which is a more rapid and non-specific inflammatory dermatitis brought about by activation of the innate immune system by the pro-inflammatory properties of chemicals or other small molecules [271].

In ACD, dendritic cells, including epidermal Langerhans cells and other dermal dendritic cell populations, are critical to sensitizing individuals to foreign antigens.

Dendritic cells are responsible for antigen capture, transport of antigens to draining lymph nodes, and activation of naïve T cells. Naïve T cells are then expanded into effector and/or memory T cell populations, which are subdivided into four main categories: TH1, TH2, TH17, and Tregs. Interferon-gamma signaling [272] polarizes T cells toward the TH1 phenotype, which is responsible for targeting and destroying intracellular pathogens. It is thought that the TH1 phenotype of T cells is the primary mediator of ACD inflammatory responses. TH1-polarized T cells can be distinguished from other T cell populations by distinct surface markers and receptors, including distinct chemokine receptors [273].

4.2.2 Hapten activation of the adaptive immune system initiates allergic contact dermatitis

Development of ACD requires the activation of T cells that have antigen-specific acquired immunity to small molecules known as haptens, or contact allergens. Haptens are common in many household and workplace materials, as well as in personal care products and jewelry. Haptens are most commonly less than 500 daltons [274, 275]; more than 100-fold smaller than the receptor-protein complexes that recognize them to activate an allergic response. Hapten is derived from Greek “to fasten,” and in order to stimulate an allergic response these small molecules must be covalently bound, or

‘fastened,’ to a protein [276]. Exceptions to the requirement of a hapten-protein covalent bond include metallic salts, such as cobalt and nickel, which instead form an ionic complex with a protein. After hapten-protein association, this complex can then be presented to T cell receptors via the major histocompatibility complex (MHC) encoded by human leukocyte antigen (HLA) genes. Peptides presented on MHC class I are recognized by CD8+ cytotoxic T cells, while peptides presented on MHC class II are recognized by CD4+ T helper cells. Because haptens can bind to peptides that are presented either in the context of MHC class I, MHC class II, or both, they have the potential to activate both cytotoxic and helper subsets of T cells.

While both patient-specific immune responses and hapten-specific features contribute to ACD, it remains unclear why some individuals, but not others, become sensitized to specific allergens. Wide genetic variation in HLA haplotypes contributes to

differential allergic responses between individuals through differing affinities of the

MHC for specific hapten-peptide pairings [277]. The amount of antigen necessary for sensitization varies considerably between different haptens, and is dependent on a variety of factors inherent to the molecule, including epidermal permeability, chemical stability, and reactivity. For example, loss-of-function mutations in the filaggrin gene that disrupt epidermal integrity increase a patient’s risk for nickel contact allergy [278].

It is important to note that hapten-peptide presentation in the context of MHC to a T cell receptor (signal 1) is not usually sufficient to activate T cells and drive the clonal expansion necessary for allergy development. A costimulatory signal (signal 2), such as activation of toll like receptor(s) (TLR), is also necessary [279]. The combination of hapten presentation on a MHC and a costimulatory signal can be sufficient for T cell activation, expansion, and development of an allergic response. TLR4 in humans binds nickel, the most common contact allergen world-wide. Interestingly, mice do not develop an allergy to nickel, which may be due to differences in non-conserved histidine residues in the mouse Tlr4 sequence that impairs nickel-binding relative to the human sequence [279]. The necessity of a costimulatory signal, such as those caused by hapten- induced activation of TLRs, explains why not all small molecules that are presented to T cells in the context of the MHC induce an allergic response.

Given the necessity for a hapten to be attached to a carrier protein to elicit an allergic response, it is not surprising that low molecular weight is a common property of

haptens. Many haptens also have lipophilic and electrophilic residues that increase affinity for covalently binding to proteins [280]. Some molecules require oxidation or other modification before they are directly reactive. For example, urushiol, the allergic component of poison ivy, is a ‘pro-hapten’ and does not stimulate an allergic response.

However, once urushiol comes in contact with the skin, urushiol is oxidized and can stimulate an immune response. Hapten stability can influence patch testing, as the active form of the allergen must be present at a sufficient concentration to elicit a measureable reaction [281].

4.2.3 T cells are critical regulators of allergic contact dermatitis

Allergic contact dermatitis is divided into three main phases: sensitization, elicitation, and resolution [282]. All of these phases are regulated in part by different populations of T cells. In the sensitization phase, haptens are collected by resident dendritic cells in the skin. Dendritic cells then migrate to the regional lymph nodes and present a hapten-peptide antigen complex to naïve T cells. Presentation of an antigen

(signal 1) and costimulation (signal 2) activates and clonally expands antigen-specific

CD4+ and CD8+ T cells. This results in the maturation and expansion of T cell population(s) that specifically recognize the hapten-protein complex in the context of

MHC. Re-exposure to the relevant hapten initiates transmigration of the expanded effector memory T cell population to the dermis and epidermis, resulting in a clinical manifestation of ACD at the sites of hapten challenge. Finally, resolution of

inflammation occurs, which is mediated in part by Treg secretion of interleukin 10 (IL-

10), which prevents extravasation of circulating effector T cells into inflamed skin [283].

In ACD, T cells cause injury to the skin via three major mechanisms: (1) producing cytokines that induce inflammation; (2) directly killing cells; and (3) activating other immune cell populations, such as NK cells and macrophages, which potentiate T-cell driven inflammatory signals and promote tissue destruction. These three pathways require the presence of effector T cells in the skin.

Following differentiation, T cells acquire the capacity to produce effector cytokines and exit the lymph nodes. Egress of T cells from the lymphatic system into the skin occurs in four main steps: (1) rolling along the vessel endothelium; (2) activation of integrins on the endothelium that slow T cell migration; (3) firm adhesion of a T cell to the endothelium (stopping the cell); and (4) transmigration, or extravasation, of a T cell from the vessel and into the skin. Reducing T cell activation and infiltration improves symptoms and reduces inflammation. This can be achieved by blocking T cell costimulatory signals or by enhancing T cell coinhibitory signals [284]. While monoclonal antibodies that reduce T cell signaling can blunt T cell-mediated responses, they are not commonly used due to cost, burden of infusion, and potential side effects.

Topical or oral steroids, the most widely used treatments for allergic contact dermatitis, broadly inhibit T cell function and are associated with a plethora of side effects. A theoretically more attractive treatment method for ACD is selectively inhibiting the

egress of activated T cells at the site of inflammation. Notably, chemokines mediate the four steps critical for T cell egress from the vasculature to the skin. Specifically targeting

T cell signals that promote activation and extravasation has the potential to treat ACD without the side effects of non-specific T cell inhibitors.

4.2.4 The chemokine receptor system as a therapeutic target for allergic contact dermatitis

The chemokine system is necessary for all aspects of ACD, from trafficking of antigen presenting cells to the lymph node to the recruitment and polarization of cells such as Treg cells and macrophages to attenuate the inflammatory response. Indeed, chemokines and chemokine receptors play a central role in inflammatory diseases, including ACD. Chemokines are small proteins that range from approximately eight to thirteen kilodaltons in mass. Chemokines are partitioned into four main families based on the relative location of cysteine within their primary structure. These families are termed XC, CC, CXC, and CX3C, respectively, where “C” denotes a cysteine and “X” refers to any amino acid [206]. Chemokines bind to and activate the chemokine receptor

(CKR) subclass of GPCRs [285]. Integrin expression, which is activated many endogenous chemokines signaling through cognate CKRs, is necessary for appropriate lymphocyte trafficking. In addition, a chemokine gradient is necessary for the transmigration of T cells from vessels and into the skin. This transendothelial migration relies on a complex process of signaling factors and protein intermediaries promoting chemokine attraction, adhesion, and motility. Chemokines regulate a variety of 88

transcription factors, including NF-kB (nuclear factor k/light-chain enhancer of activated

B cells)[286] and NFAT (nuclear factor of activated T cells) [287], which are key to T cell activation and polarization.

As GPCRs, CKRs are attractive drug targets. Despite the therapeutic potential of

CKRs, currently only three FDA approved medications target the chemokine receptor family: Maraviroc (Selzentry or Celsentri) for HIV (targeting CCR5), Plerixafor (Mozobil) for hematopoetic stem cell mobilization (CXCR4), and the recently approved mogamulizumab (Poteligeo) for mycosis fungoides and Sézary Syndrome (CCR4). It is interesting to note that despite the role of chemokines and chemokine receptors in nearly every inflammatory process, none of the chemokine receptor drugs currently approved by the FDA are for a primary inflammatory indication. One common theory about the exceptional challenge of therapeutically targeting CKRs relative to other

GPCRs is the promiscuity and redundancy of the chemokine receptor system. The chemokine receptor CXCR3, which is highly expressed in T cells found in ACD skin lesions, binds three endogenous ligands, CXCL9, CXCL10, and CXCL11. All three of these chemokines are sufficient to induce chemotaxis of CXCR3-expressing T cells [246].

Binding of multiple endogenous ligands to the same GPCR is uncommon; most GPCRs display high affinity binding to a single endogenous ligand. Adding to the complexity of chemokine receptor signaling, distinct contact allergens can activate different cellular responses that result in differential chemokine signaling patterns.

4.2.5 CXCR3 signaling in patients with allergic contact dermatitis

The CXCR3 signaling pathway, mediated by its endogenous chemokines CXCL9

(also known as monokine induced by interferon-gamma; MIG), CXCL10 (also known as interferon-induced protein 10; IP-10), and CXCL11 (interferon-inducible T-cell alpha chemoattractant; ITAC) is the most consistently upregulated chemokine signaling pathway in ACD. In both humans and mouse models, CXCR3 is upregulated in allergic contact dermatitis, but not irritant contact dermatitis [272, 288-291]. IFNg, the classic TH1- polarizing cytokine, increases expression of CXCR3 and its ligands CXCL9, CXCL10, and CXCL11. This is in contrast to other chemokines, such as CCL2, where application of either an irritant or an allergen increased transcript levels. One study examining skin lesions after patch testing found that CXCL10 was most abundant and predominantly expressed by epidermal cells (mostly keratinocytes), while CXCL9 was expressed in both the epidermis and the dermis [288]. This study demonstrated that more than 50% of infiltrating T cells in ACD expressed CXCR3, while only 20% of T cells expressed CXCR3 in ICD. Both mouse models and patients patch tested with allergens show that CXCL9 and CXCL10 are selectively upregulated in a hapten-specific manner. A separate study demonstrated that CXCL9, CXCL10, and CXCL11 were time-dependently induced in 7 out of 8 nickel-sensitized patients upon nickel sulfate exposure, with peak chemokine expression noted 48 hours after nickel elicitation [292].

4.2.6 CXCR3 signaling in murine models of allergic contact dermatitis

Differences in the chemotactic response of CD4+ and CD8+ T cell populations to

CXCR3 chemokines have also been noted in ACD models, suggesting that different signaling transducers could tune the T migratory response of specific T cell populations.

For example, CXCL10 is nearly two-fold more potent in attracting nickel-specific CD8+ T cells relative to CD4+ T cells [293], suggesting the signaling transducers downstream of

CXCR3 expressed in cytotoxic and T helper cell populations may also differ. While

CXCL9 and CXCL10 appear necessary for the recruitment of CXCR3-expressing effector memory T cells that mediate the inflammatory response in ACD, the role of CXCL11 is less clear. CXCL11 has been shown to drive regulatory T cell activity in other disease processes [211, 294], and mouse models suggest that CXCR3 expression on regulatory T cells is essential for resolution of inflammation [295]. Further studies are necessary to dissect the physiological roles of biased signaling at CXCR3 and the function of T cell subsets in ACD. Taken as a whole, these data highlight non-redundant roles for different

CXCR3 endogenous chemokines with distinct signaling pathways and provide compelling evidence that biased agonists can impact the clinical utility of drugs targeting CXCR3, and likely other CKRs.

5. Biased CXCR3 agonists differentially control chemotaxis and inflammation

The following chapter includes contributions from a number of other scientists, and originates from a manuscript currently undergoing revisions. Lowell T Nicholson, 91

Jutamas Suwanpradid, Rachel A Glenn, Nicole M Knape, Priya Alagesan, Jaimee N

Gundry, Thomas S Wehrman, Amber Reck Atwater, Michael D Gunn, Amanda S

MacLeod, and Sudarshan Rajagopal made critical contributions to the work described.

Importantly, Thomas Wehrman performed targeted phosphoprotein analysis at

PrimityBio (Fremont, CA). Amber Atwater collected skin biopsies, and Jutamas

Suwanpradid and Amanda MacLeod performed immunohistochemistry and subsequent analyses.

5.1 Identification of CXCR3 Biased Agonists

To explore the physiological relevance of biased signaling at CXCR3, we screened a panel of publically available small molecule CXCR3 ligands and identified two agonists with biased signaling properties, VUF10661 [296] and VUF11418 (Fig 20A and B). These two small molecules were previously shown to have similar affinity for

CXCR3 (33) (Table 4). We first confirmed prior reports that both compounds signal through G[alpha]i (Figure 20A) [297, 298]. However, VUF10661 significantly increased beta-arrestin-2 recruitment to CXCR3 relative to VUF11418 (Figure 20B). These small molecule CXCR3 agonists also displayed differential beta-arrestin-1 recruitment, G protein receptor kinase (GRK) recruitment, and total CXCR3 internalization (Figure 21A-

G), consistent with biased agonism between G protein and beta-arrestin signaling as discussed previously. Because bias is a relative measurement, an appropriate reference must be used. Here, we use the endogenous agonist CXCL11 as a reference, which is a

full agonist at both G protein and beta-arrestin pathways at CXCR3 [47, 299]. As expected, CXCL11 displayed increased potency for CXCR3 relative to the small molecule agonists (Figure 21). The increased efficacy in beta-arrestin recruitment with a similar degree of G protein activation by VUF10661 demonstrate that it is a beta-arrestin-biased agonist relative to VUF11418 and CXCL11. Similarly, the preserved G protein activation with lower beta-arrestin recruitment induced by VUF11418 demonstrate that it is relatively G protein-biased compared to VUF10661 and CXCL11 (Fig 20A and B, Fig 21H and I).

Figure 20: VUF10661 and VUF11418 are CXCR3 biased agonists. (A) The CXCR3 ligands VUF11418 and VUF10661 equivalently signaled through the G[alpha]i protein pathway. (B) VUF10661 recruited bta-arrestin-2 with greater efficacy but similar potency relative to VUF11418, consistent with VUF10661 being relatively beta-arrestin- biased and VUF11418 relatively G protein-biased compared to one another. (C) Stimulation of CXCR3 with VUF10661 (1 uM), but not VUF11418 (1 uM) resulted in a conformational change of a beta-arrestin-2 biosensor that monitors energy transfer between a nanoLuc donor and YFP acceptor (upper panel). CXCL11 (250 nM) is shown for reference. (D) Stimulation for 1 hour with beta-arrestin-biased agonist VUF10661 caused greater beta-arrestin-dependent CXCR3 internalization relative to VUF11418. (E) Serum response element (SRE) transcriptional activation increased relative to vehicle following treatment with a saturating concentration of VUF10661 (10 uM), but not VUF11418 (10 uM) for five hours. 10% FBS is shown as a positive control. CXCL11 (1 uM) is shown for reference. Data are normalized to the mean of 1 uM CXCL11. Panels A, B, E; *, p < 0.05; two-way ANOVA analysis, significant effect of drug. Panels C, F; one-way ANOVA; *, p < 0.05 by Tukey post hoc analysis VUF10661 to VUF11418, #, p <

0.05 by Tukey post hoc analysis relative to vehicle, corrected for 3 multiple comparisons. n=8-9 for G[alpha]i pathway experiments; n=4 for beta-arrestin-2 BRET experiments; n=15-26 wells for beta-arrestin-2 biosensor experiments (conducted over 4 independent plates); n=3-5 for internalization experiments; n=5-8 for serum response experiments.

Consistent with inducing increased beta-arrestin recruitment, VUF10661 also

induced a distinct beta-arrestin-2 conformation as assessed by an intramolecular

biosensor (Figure 20C), increased beta-arrestin-dependent receptor internalization

(Figure 20D) and promoted serum response element response factor-mediated

transcription (Figure 20E) relative to VUF11418.

Table 4: Pharmacological properties of CXCR3 biased agonists. Concentration- response data for VUF10661 and VUF11418 were fit with a 4 parameter fit equation, allowing determination of maximal response (Emax) and EC50 for G protein signaling, beta-arrestin recruitment, and CXCR3 internalization. *From Scholten et al. 2015 (33).

Ligand G[alpha]i beta- beta- beta- Total Affinity* TGF- arrestin-1 arrestin-2 arrestin-2 Internaliza pKi of [alpha] BRET BRET Internalizat tion [125I] (EC50 (M); (EC50 (M); (EC50 (M); ion (EC50 (M); CXCL11 Emax Emax (% Emax (% (EC50 (M); Emax (% (%hCXCL hCXCL11); hCXCL11); Emax (% hCXCL11); 11); Hill Hill slope; Hill slope; hCXCL11); Hill slope; slope; R2) R2) R2) Hill slope; R2) R2) 1.1E-6 1.8E-6 1.4E-6 2.5E-6 1.9E-6 VUF 60 ± 20 150 ± 10 130 ± 6 220 ± 50 140 ± 20 6.0 ± 0.1 10661 0.9 1.7 1.7 1.3 1.3 0.3 0.8 0.9 0.8 0.9 1.5E-6 3.6E-7 4.5E-7 4.3E-6 6.5E-7 VUF 110 ± 40 40 ± 3 30 ± 20 100 ± 50 90 ± 20 6.0 ± 0.1 11418 0.4 1.1 1.1 1.9 1.0 0.5 0.9 0.5 0.6 0.7

5.2 A CXCR3 beta-arrestin-biased agonist potentiates T cell- mediated inflammation and chemotaxis

After confirming ligand bias was conserved at the murine CXCR3 receptor, which has 86% sequence homology to human CXCR3 (Figure 22 A-D), we next studied how these small molecules would affect a mouse model of allergic contact hypersensitivity (CHS). As previously discussed, CHS is a model of allergic contact dermatitis that is an inflammatory condition dependent on effector memory T cells

(Figure 23A). The CHS delayed type IV hypersensitivity reaction allows an assessment of T cell-mediated inflammation and the recruitment of effector T cells to the sensitizer dinitrofluorobenzene (DNFB). Notably, topical application of the beta-arrestin-biased agonist, but not the G protein-biased agonist, potentiated the inflammatory response

(Figure 23B).

Figure 21: Additional signaling analyses of CXCR3 ligands. (A) Representative BRET titration curve for CXCR3-RLuc with beta-arrestin2-YFP, GRK2-YFP, and GRK6- YFP. HEK 293T cells were transiently expressed with CXCR3-Rluc and either GRK2- YFP, GRK6-YFP, or beta-arrestin-YFP at varying relative concentrations to determine the optimal experimental ratios and examine if constitutive association was observed in the absence of ligand stimulation. GRK6-YFP displayed a hyperbolic relationship of

YFP:CXCR3-Rluc transfection levels, (indicative of constitutive association), whereas GRK2-YFP and beta-arrestin-2-YFP displayed reduced association in the absence of ligand. Based on these data, transient transfection conditions were established to be 1 ug CXCR3-Rluc: 1 ug GRK-YFP and 1 ug CXCR3-Rluc : 2.5 ug beta-arrestin-2-Rluc. (B) HEK 293T cells transiently expressing CXCR3-Rluc and GRK2-YFP were stimulated with either vehicle, the endogenous agonist CXCL11 (500 nM) the beta-arrestin-biased agonist VUF10661 (5 uM), or the G-protein biased agonist VUF11418 (5 uM). Stimulation with the beta-arrestin-biased agonist resulted in a greater recruitment of GRK2 relative to the G protein-biased agonist as assessed by the net BRET ratio, although the G protein- biased agonist VUF11418 did result in a significant recruit GRK2 relative to vehicle. (C) Similarly, cells expressing CXCR3-Rluc and GRK6-YFP were stimulated with vehicle, CXCL11 (500 nM), VUF10661 (5 uM), or VUF11418 (5 uM). Only the beta-arrestin-biased agonist VUF10661 caused a significant increase in interaction between CXCR3 and GRK6 as assessed by the NET BRET ratio. (D) HEK 293T cells transiently expressing CXCR3-Rluc and beta-arrestin-1-YFP were stimulated with either vehicle, the beta- arrestin-biased agonist VUF10661, or the G-protein biased agonist VUF11418. Stimulation with the beta-arrestin-biased agonist for five minutes resulted in a greater recruitment of beta-arrestin-1 relative to the G protein-biased agonist as assessed by the net BRET ratio. (E) HEK 293T cells transiently expressing CXCR3-Rluc and myrpalm- mVenus, which localizes to the plasma membrane. Cells were treated with the indicated agonist for one hour as a measure of total receptor internalization, and internalization was quantified by normalization to the full endogenous agonist CXCL11 (1 uM). Both agonists caused internalization, with the beta-arrestin-biased agonist having a statistically greater Emax relative to G protein-biased agonist. (F) Serum response element-response factor (SRF), a transcription factor similar but distinct from SRE, transcriptional activation was assessed by a luciferase reporter and was increased relative to vehicle following treatment with a saturating concentration of the beta- arrestin-biased agonist VUF10661 (10 uM), but not the G protein-biased agonist VUF11418 (10 uM), after four-hour stimulation. Reporter activity following 10% FBS is shown for reference. (G) Utilizing the G protein activity TGF-[alpha] shedding assay, cells devoid of G protein subunits were transfected with the empty vector pcDNA3.1 (as opposed to CXCR3), along with the G[alpha]i subunit and AP-TGF-[alpha] to test for off target activity. Unlike cells transfected with CXCR3, no appreciable normalized responses were observed. (H) G protein signal induced by the biased agonists VUF10661 and VUF11418 plotted alongside the G protein signal from the more potent endogenous ligand human CXCL11. (I) Beta-arrestin-2 signal induced by the biased agonists VUF10661 and VUF11418 plotted alongside the beta-arrestin-2 signal from the more potent endogenous ligand human CXCL11. For all panels, CXCL11 is shown for reference and not included in statistical analyses. Panels B,C,F, one-way ANOVA; *, p < 0.05 by Tukey post hoc analysis of vehicle to drug treatment; Panel D,E, p < 0.05; two-

way ANOVA analysis. Panel A is representative of three separate experiments; for GRK BRET recruitment, n=6-8; for beta-arrestin-1 BRET recruitment, n=3-5; for CXCR3 total internalization, n=4-6; for serum response experiments, n=5-8; for empty vector negative control G[alpha]i experiments n=6-8; for G[alpha]i pathway experiments n=6-9; for beta- arrestin-2 BRET n=4, n.s.; not significant.

Figure 22: Biased signaling is conserved at murine CXCR3. (A) Homology summary from NCBI comparing human (hCXCR3) and murine (mCXCR3) CXCR3 receptors and ligands. The CXCR3 signaling pathway displays a high degree of conservation between species. (B) NCBI alignment of human and murine CXCR3. (C) Similar to the human receptor, the CXCR3 ligands VUF11418 and VUF10661 equivalently signal through the G[alpha]i protein pathway at mCXCR3. (D) Similar to the human receptor, divergent beta-arrestin-2 recruitment was observed at mCXCR3. The more potent endogenous ligand murine CXCL11 is shown for reference in both panel C and panel D. *, p < 0.05; two-way ANOVA analysis, significant effect of drug. n=3-6 replicates per group. n.s.; not significant. Experiments were conducted in HEK 293T cells.

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The potentiation induced by VUF10661 was not observed in either beta-arrestin-2 knockout mice (Figure 23C) or CXCR3 knockout mice (Figure 24), consistent with the effects of VUF10661 requiring both CXCR3 and beta-arrestin-2, the predominant arrestin isoform in T cells (23).

Figure 23: A beta-arrestin-biased, but not G protein-biased, CXCR3 agonist increases inflammation and chemotaxis of effector/memory T cells. (A) Experimental design of the DNFB contact hypersensitivity model of inflammation. DNFB sensitization

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was induced with 0.5% DNFB and contact allergy was elicited five days later with 0.3% DNFB. (B) Topical application of the beta-arrestin-biased agonist VUF10661 on the ear of wild-type mice after DNFB elicitation caused a potentiation of ear thickness compared to either vehicle or the G-biased agonist VUF11418. (C) The beta-arrestin-biased agonist VUF10661 did not potentiate ear thickness in beta-arrestin-2 KO mice. (D) In the absence of DNFB-induced expansion of effector T cells, neither VUF10661 nor VUF11418 application increased inflammation in vehicle control ears. (E) The beta-arrestin-biased agonist VUF10661 resulted in greater migration of effector T cells compared to G-biased agonist VUF11418. VUF11418 (1 uM) did not cause significant chemotaxis above 0 nM (p < 0.05, two –tailed t-test). Chemotaxis of beta-arrestin-2 KO effector T cells was severely attenuated and right shifted, while eliminated in effector T cells from CXCR3 KO mice. (F) The beta-arrestin-biased agonist VUF10661 increased infiltration of skin effector T cells to DNFB allergen elicited ears relative to vehicle or G protein-biased agonist VUF11418 treatment. (G) The beta-arrestin-biased agonist VUF10661 induced increase in skin effector T cells was decreased in beta-arrestin-2 KO mice. Panel B; *, p < 0.05; two- way ANOVA analysis, significant effect of drug. Panel E; *, p < 0.05; two-way ANOVA analysis, significant effect of drug for WT VUF10661 vs WT VUF11418 (Tukey post hoc analysis for 1 uM also p < 0.05); significant effect of genotype for WT VUF10661 vs beta- arrestin-2 KO VUF10661; significant effect of genotype for WT VUF10661 vs CXCR3 KO VUF10661. For CHS experiments, n=7-11 per group; for transwell migration assays, n=3- 4 per drug treatment, and the p value is corrected for the six comparisons noted in the panel. n.s.; not significant.

Figure 24: The inflammatory effects of the CXCR3 beta-arrestin-biased agonist VUF10661 were absent in CXCR3 KO mice. VUF10661 and VUF11418 did not significantly alter the ear thickness of CXCR3 knockout mice undergoing DNFB CHS paradigm, and in absence of DNFB elicitation the ligands did not cause an increase in 102

ear thickness. CXCR3 knockout mice treated with VUF10661, VUF11418, or vehicle did not show a statistically different difference in ear thickness. Two-way ANOVA, no significant effect of drug in either panel. n=5-7 mice per treatment group. n.s.; not significant.

Importantly, the small molecules did not cause inflammation in the absence of DNFB- induced T-cell allergy and therefore did not act as irritants or haptens (Figure 23D).

Given the established role of CXCR3 in T cell chemotaxis [209] and the higher expression of CXCR3 on CD8+ cells relative to CD4+ cells in allergic contact dermatitis [293], we hypothesized that the increased inflammation induced by the beta-arrestin-biased agonist was due to increased effector memory CD8+ T cell chemotaxis compared to a G protein-biased agonist. Supporting this, the beta-arrestin-biased agonist increased chemotaxis relative to the G protein-biased agonist in effector memory T cells from wild- type mice in a transwell migration assay (Figure 23E). This difference in migration was eliminated in activated CD8+ T cells from beta-arrestin-2 knockout mice (Figure 23E) and reduced in activated CD4+ T cells (Figure 25). Consistent with the role of beta- arrestin mediating chemotaxis at other CKRs [214, 300, 301], migration of effector memory T cells to the endogenous chemokine murine CXCL10 was also severely attenuated in beta-arrestin-2 KO mice (Figure 25B-D). No migration was observed in T cells from CXCR3 KO mice to either the beta-arrestin-biased or the G protein-biased agonist (Figure 25E).

With the relative differences in biased agonist-induced chemotaxis, we then investigated if topical application of the biased agonists caused an increase in skin

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effector memory T cells. The beta-arrestin-biased agonist increased the number of ear effector memory cytotoxic T cells relative to both vehicle and the G protein-biased agonist treatment following DNFB elicitation in wild-type mice (Figure 23F).

Figure 25: Beta-arrestin-2 knockout attenuated chemotaxis to the endogenous chemokine murine CXCL10, and both VUF10661 and murine CXCL10 only induced significant chemotaxis in CD44+ T cell populations. (A) Similar to CD8+CD44+ T cells, the beta-arrestin-biased agonist VUF10661 caused greater migration of CD4+CD44+ T cells compared to the G protein-biased agonist VUF11418. Beta-arrestin-2 knockout mice displayed a right shift in migration towards VUF10661 relative to WT mice, and no migration to either agonist was observed in T cells isolated from CXCR3 KO mice. (B) CD4+CD44+ and (C) CD8+CD44+ T cells isolated from WT mice displayed greater maximal chemotactic migration towards murine CXCL10 relative to T cells isolated from beta-arrestin-2 KO mice. No migration towards murine CXCL10 was observed in T cells 104

isolated from CXCR3 KO mice. Unlike CD44+ cells, CD44- T cells did not display differences in migration to either (D) the endogenous ligand murine CXCL10 or (E) the small molecule biased CXCR3 agonists. *, p < 0.05; two-way ANOVA analysis, significant effect of drug for noted comparisons in CD44+ synthetic ligand groups, and significant effect of genotype between all treatment groups in CD44+ murine CXCL10 treatment groups. In CD44- cells, two-way ANOVA analysis demonstrates no significant effect of either drug or genotype in either synthetic ligand groups or of genotype in murine CXCL10 treated groups. n=3-5 mice per treatment group.

Treatment with the beta-arrestin-biased agonist VUF10661 also increased ear effector memory T helper cells relative to G-biased agonist treatment (Figure 26 A and

B). The ears of beta-arrestin-2 KO mice accumulated fewer effector memory T cells compared to wild-type mice (Figure 23G and Figure 26C). No change in the regulatory T cell frequency of the CD4+ population was observed (Figure 26 D and E). Together, these findings are consistent with the beta-arrestin-biased agonist increasing skin inflammation by promoting effector memory T-cell chemotaxis and recruitment.

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Figure 26: Biased CXCR3 ligands differentially increased CD4+CD44+ and total T cells to DNFB-treated ears. (A) Wild-type mice topically treated with the CXCR3 beta-arrestin-biased agonist VUF10661 (50 uM topical) displayed an increase in total CD4+CD44+ cells relative to the G protein-biased agonist VUF11418 (50 uM topical). (B) Total CD3+ cells were also increased in the VUF10661 treatment group relative to the VUF11418 treatment group. (C) Beta-arrestin-2 KO mice undergoing DNFB ear elicitation and subsequently treated with vehicle (as opposed to biased CXCR3 agonists) displayed reduced ear CD8+CD44+ T cell infiltrate relative to wild-type mice. (D) Mice displayed no significant difference in regulatory T cell recruitment (as measured by CD4+Foxp3+) after biased agonist treatment as assessed by the relative proportion of regulatory T cells. (E) The relative abundance of CD4+Foxp3+ compared to total CD4+ cells demonstrated in the contour plots. Panels A and B, one-way ANOVA; *, p < 0.05 by Tukey post hoc analysis; Panel C, *, p < 0.05 unpaired two-tailed test. n=3-9 mice per treatment group. Contour plots are representative of three mice.

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5.3 Patients positive for contact hypersensitivity coexpress CXCR3 and beta-arrestin in T cells in the skin

A major confounder in studies of chemokine biology is differences in expression patterns and function between species [302]. To correlate CXCR3 signaling with human contact hypersensitivity, we sampled skin from patch-tested patients (Figure 27A). Skin patch testing involves the placement of contact allergens directly on the skin. Positive patch tests reactions include elevated erythematic plaques, with severe reactions ulcerating. Patch testing is the gold standard for diagnosis of allergic contact dermatitis and the role of T cells in the allergic contact hypersensitivity pathophysiology that underlies this disease is well established [303]. Ninety-six to 120 hours after allergen application, the patch test was read, and biopsies of positive and negative sites were collected and stained for the T cell marker CD3, CXCR3, and beta-arrestin (Figure 27B).

More T cells and more T cells coexpressing CXCR3 and beta-arrestin were observed in

ACD biopsies relative to non-lesional (negative) biopsies at the epidermal-dermal junction (Figure 27 C and D). In addition, the relative percentage of T cells coexpressing these markers was increased in ACD biopsies (92/189, or 49%) relative to non-lesional biopsies (8/23, or 35%). To test if beta-arrestin signaling and G protein-biased signaling resulted in a similar effect on human T cell migration, we isolated leukocytes from peripheral blood and tested chemotaxis to the CXCR3 biased agonists. Consistent with our observations in mice, the beta-arrestin-biased agonist caused increased human effector memory T cell migration relative to the G protein-biased agonist in human T

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cells (Figure 27 E and F, and Figure 28). These findings are consistent with a similar role for CXCR3-mediated signaling through beta-arrestins in T cells in human CHS responses.

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dermatitis patch testing. Colored regions on day 5 signify a positive response. (B) Representative immunohistochemistry of CXCR3 (green), CD3 (red), beta-arrestin (purple), and Hoechst (blue) in skin from allergen patch-tested skin and matched non- lesional (NL) controls from the same patient. Data are representative of 3 patient samples with similar results, original magnification x200 (left) and x400 with scale bar 100 um and 50 um, respectively. The white boxes in x200 images indicate the area of magnified images in subsequent pictures and are located at the epidermal (epi)-dermal junction. In allergen patch-tested skin, this area was where we found most T-cell clusters as previously described (69) whereas the same area in NL skin was devoid of such immune cell conglomerates and served as a control. (C) Quantitative analysis of dermal CD3+ T cells and (D) co-expression of CD3 with CXCR3 and beta-arrestin in skin from allergen patch-tested skin (ACD) and matched non-lesional (NL) controls from the same patient. The beta-arrestin-biased agonist VUF10661 resulted in greater migration than the G-biased VUF11418 in (E) CXCR3+ CD8+ and (F) CD44+CD8+ in T cells from patch tested patients. Patch test quantitative analyses are expressed as positive cells ± SEM from 6 microscopic fields at 400×, from a total of three patients. For panels C and D, *, p < 0.05; unpaired two-tailed t-test. For panels E and F, *, p < 0.05; two-way ANOVA, significant effect of drug. Histology and quantification were conducted in the laboratory of Dr. Amanda MacLeod.

Figure 28: Human T cell chemotaxis. Human T cell chemotaxis towards CXCR3 biased agonists. In addition to the significant difference in chemotaxis of CD8+CD44+ T

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cells isolated from the peripheral blood of patients, total (A) T cells (n=5) and (B) CD4+CD44+ T cells (n=5) demonstrated increased migration towards the beta-arrestin- biased agonist VUF10661 relative to the G protein-biased agonist VUF11418. No significant difference was observed between the agonists for (C) CD4+CXCR3+ T cells (n=3). *, p < 0.05; two-way ANOVA analysis, significant effect of drug; n.s., not significant.

5.4 beta-arrestins activate Akt and regulate T cell chemotaxis.

Given the overlap of signaling pathways mediated by both beta-arrestins and G proteins, it was unclear which specific signaling effectors downstream of beta-arrestins were responsible for differences in chemotaxis and inflammation induced by beta- arrestin- and G protein-biased agonists. To identify potential pathways, we performed a targeted flow cytometry-based analysis of intracellular phosphorylation in human peripheral blood mononuclear cells (PBMCs) following stimulation with vehicle,

VUF10661, or VUF11418. The Akt pathway, known to be regulated by beta-arrestin, displayed qualitative differences in these targeted analyses. The beta-arrestin-biased agonist increased phosphorylation of Akt residues Threonine 308 and Serine 473, which are necessary for full efficacy kinase activity, relative to the G protein-biased agonist

(Figure 29A). Differences in phosphorylation of Serine 9 on GSK3-beta, a position known to inhibit GSK3-beta kinase activity and regulated in part by Akt, were also observed (Figure 29A). We verified these differences in Akt phosphorylation in a T cell line (Jurkat) stably expressing CXCR3, observing greater Akt Thr 308 phosphorylation by the beta-arrestin-biased VUF10661 relative to the G protein-biased VUF11418 (Figure

29B). Greater amounts of phospho-Akt Thr 308 could be coimmunoprecipitated with

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beta-arrestin-2 following CXCR3 activation with VUF10661 compared to either

VUF11418 or vehicle (Figure 29C).

Figure 29: Akt activation is dependent on beta-arrestin-2 and promotes CXCR3-mediated chemotaxis. (A) Selected heat map of targeted analysis of intracellular phosphorylation (log2 fold change relative to vehicle) of human CD8+ T cells after stimulation with either VUF10661 (1 uM) or VUF11418 (1 uM) normalized to vehicle

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demonstrating agonist-dependent differences in GSK3-beta/Akt pathway signaling. Increased phosphorylation of Akt activating sites 308 and 473 and decreased phosphorylation of the GSK3-beta inhibiting site 9 was observed following VUF10661 stimulation. (B) Akt Thr 308 phosphorylation in Jurkat cells (T cell line) stably expressing CXCR3 was higher after stimulation with the beta-arrestin-biased agonist VUF10661 compared to a G protein-biased agonist VUF11418 at 60 minutes. (C) Quantification of co-immunoprecipitation of pAkt Thr 308 with FLAG-beta-arrestin-2 following 60 minute agonist stimulation (1 uM). (D) Representative co- immunoprecipitation and immunoblots of FLAG-beta-arrestin-2 followed by immunoblot for pAkt 308 following 60 minute agonist stimulation. (E) Quantification of phosphorylated Akt 308 demonstrated that siRNA knockdown of beta-arrestin-2 eliminated Akt Thr 308 phosphorylation following 60 minute stimulation with VUF10661 (1 uM) (F) Representative immunoblot of siRNA knockdown data from panel E (G) Akt inhibition decreases T cell chemotaxis. Pretreatment of leukocytes isolated from the spleens of WT mice with the selective Akt inhibitor AZD5363 (100 nM) decreased effector T cell chemotaxis to VUF10661 (1 uM). Inset demonstrates a paired comparison between vehicle and AZD5363 conditions. For panel B, *, p < 0.05, unpaired two-tailed t-test; for panel C *, p < 0.05, one-way ANOVA followed by Tukey post hoc comparison, # p <0.05, significantly greater than vehicle by one-way ANOVA followed by Tukey post hoc comparison; for panel E, *, p < 0.05, two-way ANOVA, significant interaction of siRNA and ligand treatment followed by Tukey post hoc comparison (corrected for six multiple comparisons) of control siRNA-VUF10661 vs. beta-arrestin-2 siRNA-VUF10661; for Panel G,*, p < 0.05; repeated measures two-way ANOVA, significant interaction of AZD5363 and VUF10661 followed by Tukey post hoc comparison (corrected for six multiple comparisons) of Vehicle-VUF10661 vs. AZD5363- VUF10661; p < 0.05; Inset of panel G, p < 0.05, paired two-tailed t-test. For immunoblot experiments, n=4-6 per condition; for immunoprecipitation image is representative of 3 independent experiments, for migration experiment, n=8 mice. Cellular experiments in panels C-F were conducted in HEK 293T cells. NT, non-transfected control; Veh, vehicle.

siRNA knockdown of beta-arrestin-2 abolished Akt Thr 308 phosphorylation induced by VUF10661 stimulation (Figure 29, E and F) and attenuated Akt Ser 473 phosphorylation (Figure 30). VUF10661 and VUF11418 did not demonstrate differential phosphorylation of ERK (Figure 30), a kinase whose activity has frequently been shown to be regulated by both G proteins and beta-arrestins [239, 304].

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Figure 30: Diverging Akt, but not ERK 1/2, phosphorylation following stimulation with VUF10661 or VUF11418 in a T cell line stably expressing CXCR3. (A) Representative immunoblot of phospho-Akt Thr 308 in Jurkat cells following stimulation with beta-arrestin-biased VUF10661 (1 uM) or G protein-biased VUF11418 (1 uM) for the indicated duration, demonstrating greater Akt 308 phosphorylation relative to total Akt at 60 minutes, but not earlier time points. (B) Representative immunoblot of phospho-ERK1/2 in Jurkat cells following stimulation with beta-arrestin-biased VUF10661 (1 uM) or G protein-biased VUF11418 (1 uM) for the indicated duration demonstrating no observed difference in phosphorylation relative to total ERK signal at any time point tested. (C) Representative immunoblot of phospho-Akt Ser 473 signal in 113

HEK 293N cells treated with either control siRNA or siRNA targeting beta-arrestin-2 and subsequently stimulated with either vehicle or VUF10661 (1 uM). Similar to phospho-Akt Thr 308, siRNA knockdown of beta-arrestin-2 attenuated phospho-Akt Ser 473 signal. (D) siRNA knockdown of beta-arrestin-2 was confirmed, with relative knockdown ranging from 67%-85% relative to control siRNA. (E) Quantification of phospho-ERK signal in Jurkat cells as calculated by phospho-ERK/total-ERK percent above vehicle. No difference in ERK phosphorylation between treatment groups was observed at 5, 30, or 60 minutes. (F) Quantification of phospho-Akt Thr 308 signal in Jurkat cells as calculated by phospho-Akt/total Akt, normalized to vehicle treatment. A significant difference in Akt phosphorylation was observed at 60 minutes, but neither 5 nor 30-minute time points. (G) Quantification of phospho-Akt Thr 473 signal in Jurkat cells. Similar to phospho-Akt 308 signal, a significant difference in Akt phosphorylation was observed at 60 minutes, but neither 5 nor 30 minute time points. (H) Quantification of phospho-Akt 473 signal following siRNA knockdown. Panels E, F, * p < 0.05, two-way ANOVA, Bonferroni post hoc comparison significant at 60 minute, but neither 5 nor 30 minute, time points. For panel H, * p < 0.05, two-way ANOVA, main effect of both siRNA and of ligand treatment. n=3-8 replicates per condition, immunoblots are representative of at least 3 independent experiments.

Consistent with specific beta-arrestin-mediated Akt activation contributing to

chemotaxis, pretreatment of lymphocytes with the Akt inhibitor AZD5363 decreased

effector memory T cell chemotaxis towards VUF10661 (Figure 29G). T cell chemotaxis was also inhibited by either the PI3K inhibitor LY20042 (Figure 31 A and B) or pertussis

toxin (Figure 31 C and D). These findings are consistent with both pertussis toxin-

sensitive G protein signaling and beta-arrestin-mediated Akt signaling contributing to

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CXCR3-mediated chemotaxis.

Figure 31: Both pertussis toxin and PI3K inhibition eliminated effector T-cell migration to VUF10661. Pretreatment of splenocytes isolated from wild-type mice for 1 hour with the PI3K inhibitor LY294002 (100 uM) eliminated (A) CD8+CD44+ and (B) CD4+CD44+ cell migration towards VUF10661 (1 uM). Pretreatment of splenocytes isolated from wild-type mice for 1 hour with pertussis toxin (100 ng) eliminated (C) CD8+CD44+ and (D) CD4+CD44+ cell migration towards VUF10661 (1 uM). *, p < 0.05 repeated measures two-way ANOVA followed by Tukey post hoc comparison (corrected for six multiple comparisons) of vehicle-VUF10661 to inhibitor-VUF10661 and vehicle-VUF10661 to vehicle-vehicle. n=5-6 mice per group. PTX, pertussis toxin.

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6. Discussion

In chapter 5, we demonstrate that small molecule biased CXCR3 agonists with similar receptor affinity but divergent G protein and beta-arrestin efficacies cause distinct physiological effects on T cell-mediated skin inflammation through differential T cell chemotaxis. These data are consistent with the data reported in chapter 3 and discussed in section 3.10, where endogenous chemokines of CXCR3 initiate distinct G protein and beta-arrestin signaling patterns.

6.1 Implications for CXCR3 Drug Design

We found that a small molecule beta-arrestin-biased agonist (VUF10661) was superior in promoting T cell chemotaxis compared to a G protein-biased agonist

(VUF11418). Similar to prior studies, we found that both G protein and beta-arrestin signaling were necessary for CKR-mediated chemotaxis [214, 241]. These findings suggest that biased endogenous chemokines promote distinct physiological effects, with clear relevance for drug discovery at CXCR3 and other chemokine receptors. For example, drugs designed to block CXCR3+ T cell movement to reduce inflammation ought to impair beta-arrestin signaling, while drugs used to increase CXCR3+ T cell recruitment, e.g., in cancer immunotherapy, should promote signaling through beta- arrestin and G[alpha]i. The distinct physiological effects observed with biased agonists offer a plausible explanation for the difficulties in drugging chemokine receptors and

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also for biased signaling patterns encoded by different endogenous chemokines at the same receptor.

Given the roles of beta-arrestin in the ‘negative feedback’ of receptor signaling

(desensitization and internalization), it was unclear how small molecules regulating complex G protein and beta-arrestin signaling pathways would interact in physiologically relevant models of chemotaxis and inflammation. Our findings show that CXCR3 biased agonists can differentially impact inflammatory processes. Beta- arrestins scaffold a number of effectors that control cell polarity and influence cellular migration [305-308]; however, it was unclear which of these effectors were relevant to chemotaxis. This work implicates Akt, regulated by beta-arrestin, as being critical for full efficacy cell migration induced by CXCR3 signaling.

6.2 Beta-arrestin / Akt regulation of T cell chemotaxis

Using targeted analysis of intracellular phosphorylation followed by hypothesis- driven experiments, we identified phosphorylation of Akt, which is regulated by beta- arrestin, as necessary for full chemotactic function. Although pertussis toxin eliminated T cell chemotaxis, Akt inhibition did not fully inhibit migration, suggesting that multiple

CXCR3-mediated pathways mediate T cell chemotaxis. Notably, Akt has a well- established role in mediating cell polarity and migration. Akt translocates to the leading edge of migrating cells [309, 310], and this pathway is known to be regulated by endogenous CXCR3 chemokines [211, 311]. Moreover, beta-arrestins have been shown

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to mediate Akt activation via changes in phosphorylation through the formation of signaling complexes downstream of the D2 dopamine receptor [13, 312] and the insulin receptor [313]. The G protein-biased agonist failed to induce significant chemotaxis above baseline, and at high concentrations reduced migration below basal levels, which could be due to reduced Akt phosphorylation or off-target effects at high drug concentrations.

Despite prior implications of both beta-arrestin and G protein signaling as critical to cell function and migration downstream of other chemokine receptors, such as

CXCR4 [214, 241, 314, 315], it was unclear how biased agonists would differentially impact physiological models of T cell movement and disease, if at all. Despite the fact that the beta-arrestin-biased agonist caused greater CXCR3 internalization compared to the G protein-biased agonist, the beta-arrestin-biased agonist increased chemotaxis and potentiated a T cell-mediated inflammatory response. Consistent with other chemokine receptors [262, 316], elimination of CXCR3-mediated chemotaxis with pertussis toxin towards the beta-arrestin-biased agonist implicates both G proteins as well as beta- arrestins in chemotaxis. Recent studies classify CXCL11 as a beta-arrestin-biased ligand relative to the other two CXCR3 endogenous ligands CXCL9 and CXCL10 [213, 299].

Interestingly, CXCL10 and CXCL11 bind non-overlapping region(s) of CXCR3. In addition, CXCL11 binds to both G protein coupled and pertussis toxin uncoupled forms of CXCR3. In contrast, the relatively G biased endogenous ligand CXCL10 displayed no

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affinity for the pertussis toxin uncoupled form of CXCR3 [317], providing an illustrative example of multisite chemokine binding observed at CXCR3 [318]. These data suggest that a different transducer, such as beta-arrestin, may stabilize a distinct active receptor conformation when bound to CXCL11. Functionally, CXCL11 has previously been shown to induce greater chemotactic response relative to CXCL9 and CXCL10 [246, 252], and suggests that CXCL11 may be the most ‘proinflammatory’ of the endogenous

CXCR3 ligands. Consistent with beta-arrestin playing a central role in T cell migration, we found that a beta-arrestin-biased agonist increased CXCR3-mediated T cell chemotaxis and increased a T cell-mediated inflammatory response relative to a G protein-biased agonist. In addition, T lymphocytes isolated from beta-arrestin-2 KO mice displayed defective chemotaxis to an endogenous CXCR3 chemokine as evidenced by a

~10-fold decrease in potency, consistent with prior observations [241]. Our data are consistent with a model in which activating beta-arrestin signaling can increase T cell migration and function, while inhibiting either beta-arrestin signaling or beta-arrestin association with Akt would oppose migration and inflammation.

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7. Materials and methods

This chapter summarizes the major experimental materials and methods utilized throughout the experiments presented in this dissertation.

7.1.1 Cell Culture

HEK293 cells stably expressing a modified firefly luciferase enzyme linked to the cyclic AMP (cAMP) sensor Epac (Glosensor, Promega) active in the presence of cAMP and Luciferin (Promega), were used to quantify G[alpha]i signaling with the glosensor assay. HEK293T cells were used for bioluminescence resonance energy transfer (BRET) experiments to assess beta-arrestin recruitment. The cells were maintained in Minimum

Essential Media (MEM) (Corning) containing 1% penicillin/streptomycin and 10% Fetal

Bovine Serum (FBS) (Corning). Cells were grown at 37°C in a humidified 5% CO2 incubator in poly-d-lysine coated tissue culture plates. Media was changed every 48-72 hours.

7.1.2 Transfections

Luciferase attached to the C-terminus of CXCR3A and CXCR3B was used in donor constructs for BRET assays. Luciferase constructs were encoded either in a Renilla luciferase (Rluc) pN-3 vector (Promega) or Nanoluc Luciferase vector pNL1.1

(Promega). YFP was attached to the C-terminus of beta-arrestin-1/2; YFP tags for beta- arrestins and GRKs were used as acceptors in BRET assays. Transient transfections were conducted with calcium phosphate for all BRET assays as previously described [319].

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Briefly, calcium phosphate transfections were conducted with HEPES buffered saline with 4 µg of the CXCR3-RLuc receptor, 10 ug of beta-arrestin-2-YFP, 10 ug beta-arrestin-

1-YFP, 4 ug of GRK2-YFP, or 4 ug of GRK6-YFP and 0.25M CaCl2. 50 ng of the Nanoluc beta-arrestin-2 biosensor was determined to be the optimal concentration for conformational assessment, and transfected with untagged receptor (4 ug CXCR3A, 5 ug

CXCR3B) for biosensor studies.

7.1.3 Bioluminescence Resonance Energy Transfer (BRET)

Twenty-four hours after transfection, cells were plated onto a 96-well plate at

50,000-100,000 cells/well. Approximately 44 hours after transfection, media was changed to MEM (Corning) supplemented with 10mM HEPES, 0.1% bovine serum albumin and

1% penicillin/streptomycin. After approximately three to four hours of serum starvation, cells were washed with room temperature PBS. Next, 80 µL of a coelenterazine-h/HBSS solution (3 uM coelenterazine-h) was added. Ligands were prepared at 5x concentration and read by a Mithras LB940 instrument (Berthold) with

485nm and 530nm emission filters. The BRET ratio was calculated using Net BRET ratio

= [YFP emission at 530nm] / [Rluc emission at 485 nm] -cf, where cf represents the BRET ratio in the vehicle control group. For the bystander BRET total internalization assay, titration response experiments determined that 400ng of myr-palm-mVenus and 2.5 ug of 2xFYVE-mVenus and a constant 4 ug of Rluc labeled CXCR3 maximized signal:noise, and these concentrations were used throughout for BRET internalization experiments.

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For CXCR3-beta-arr interaction, the net BRET ratio was quantified five minutes following ligand addition. For bystander BRET-based receptor internalization assays

(receptor association with FYVE-mVenus or dissociation from a myristoyl - palmitoylated-mVenus (myr-palm)), cells were not serum starved and instead incubated in assay buffer with ligand for one hour before adding coelenterazine-h as described above. Recombinant human CXCL4, CXCL9, CXCL10, and CXCL11 were obtained from either Peprotech (Rocky Hill, NJ) or BioLegend (San Diego, CA).

7.1.4 GloSensor cAMP Assay

Lipofectamine 2000 (Thermo Fisher) transfected cells were plated on 96-well plates 24 hours after transfection with either CXCR3A or CXCR3B at a density of 10-

25,000 cells/well. At approximately 44 hours post transfection, cells were serum starved for two hours. Next, the plate was washed with room temperature PBS, and 60 µL of

GloSensor cAMP Reagent (Promega) in HBSS with 20 mM HEPES (Gibco) was then added per well. The plate was incubated for two hours in the dark at room temperature.

For G[alpha]i pathway assay, the cells were then stimulated with 100 nM isoproterenol, incubated for five minutes, and luminescence over one second was quantified by a

Mithras LB940 instrument. Vehicle or CXCR3 ligands were then added, incubated for ten minutes, and the plate read a second time. Data were normalized to vehicle treated wells, and to 1 uM CXCL11 for G[alpha]i studies. For G[alpha]s studies, baseline luminescence

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was determined prior to the addition of CXCR3 ligand, and the plate read a second time after ligand addition.

7.1.5 TGF-alpha shedding assay

Directed CXCR3 G[alpha]i activity was assessed by the transforming growth factor-alpha (TGF-alpha) shedding assay as previously described [199]. Briefly, HEK 293 cells lacking G[alpha]q, G[alpha]i, G[alpha]s, and G[alpha]12/13 were transiently transfected with CXCR3, modified TGF-[alpha]-containing alkaline phosphatase (AP-

TGF-[alpha]), and either the G[alpha]q/i1,2 assay subunit (as the G[alpha]i2 is observed to be indispensable in CXCR3 T cell signaling [320]) or a negative control ∆"C" subunit

(lacking G[alpha] distal amino acids to preclude receptor interaction) and were reseeded twenty-four hours later in Hanks’ Balanced Salt Solution (HBSS) (Gibco, Gaithersburg,

MD) supplemented with 5mM HEPES in a Costar 96-well plate (Corning Inc., Corning,

NY). Cells were then stimulated with the indicated concentration of ligand for one hour.

Conditioned media (CM) containing the shed AP-TGF-alpha was transferred to a new

96-well plate. Both the cell and CM plates were treated with para-nitrophenylphosphate

(p-NPP, 100 mM) (Sigma-Aldrich, St. Louis, MO) substrate for one hour, which is converted to para-nitrophenol (p-NP) by AP-TGF-alpha. This activity was measured at

OD405 in a Synergy Neo2 Hybrid Multi-Mode (BioTek, Winooski, VT) plate reader immediately after p-NPP addition and after one-hour incubation.

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7.1.6 Small molecules and peptides

VUF10661 (Sigma-Aldrich), VUF11418 (Aobious), LY294002 (Sigma-Aldrich),

AZD5363 (Axon Med Chem) were dissolved in DMSO make stock solutions and stored at -20°C in a desiccator cabinet. Recombinant human and murine CXCL4, CXCL9,

CXCL10, and CXCL11 (Peprotech) were diluted according to manufacturer specifications and aliquots were stored at -80°C until use.

7.2 Generation of FLAG-tagged CXCR3 and C-terminal Truncation Mutants

FLAG-tagged CXCR3 isoform constructs were created by PCR amplification with the signal sequence hemagglutinin (HA) 5’ to the FLAG-epitope sequence as previously described [321]. The PCR product was gel purified and inserted into pcDNA3.1 using

XbaI endonuclease cleavage sequence 5’ to the HA sequence and an EcoRI endonuclease cleavage sequence 3’ to the stop codon. L344X and L391X C-terminal CXCR3 truncation mutants were created by PCR amplification of WT CXCR3A or CXCR3B, introduction of a HindIII endonuclease cleavage sequence 5’ to the start codon on the forward primers

(CXCR3A: 5’-CGCGGTAAGCTTATGGTCCTTGAG-3’; CXCR3B: 5’-

CGGACCGAAGCTTATGGAGTTGAG-3’) and the introduction of a KpnI endonuclease cleavage sequence 3’ to either the 343rd or 390th amino acid on the reverse primer (5’-

ACCCATGGTACCATCCCTCTCTGG-3’). The amplified PCR product was gel purified and inserted in the pRluc-N3 expression vector. All constructs were verified by Sanger

DNA sequencing. 124

7.3.1 GRK knockdown

For siRNA GRK knockdown studies, HEK 293T cells were transfected with

Lipofectamine 3000 (Thermo Fisher) per manufacturer specifications within a 96 well plate with 5ng of either CXCR3A-Rluc or CXCR3B-Rluc, 12.5ng of beta-arr2-YFP, and

30ng GRK2/GRK3 siRNA (60ng total), or 30ng GRK5/6 siRNA, or 60ng control siRNA per well. Cells were then stimulated with 500 nM of CXCL11 for 5 minutes, and the

BRET signal of beta-arrestin recruitment recorded. GRK2, GRK3, GRK5, GRK6, and control siRNA sequences were used as previously validated and described [60].

7.3.2 DiscoverX Active Internalization Assay

This assay was conducted as previously described [213] and in accordance with manufacturer protocols. Briefly, an Enzyme Acceptor-tagged beta-arrestin and a

ProLink tag localized to endosomes are stably expressed in U2OS cells. Untagged

CXCR3A or CXCR3B was transiently expressed. Beta-arrestin-mediated internalization results in the complementation of the two beta-galactosidase enzyme fragments that hydrolyze a substrate (DiscoveRx) to produce a chemiluminescent signal.

7.3.3 SRE/SRF pathway assay

293T cells were transiently transfected with SRE or SRF and either unlabeled

CXCR3A (4 ug) or CXCR3B (5 ug). 4 hours after transfection, the cells were plated on a

96 well plate at a concentration of 25,000 cells/well. 24 hours after transfection, cells were

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serum starved overnight. The next day, cells were incubated with ligand for five hours and subsequently lysed with passive lysis buffer (Promega) for ten minutes as previously described [322]. Luciferin was added to the lysate and luminescence was quantified using a Mithras LB940 instrument with no wavelength filter between the cells and the photomultiplier as previously described [319]. To observe MEK dependent effects of CXCL11, the MEK inhibitor PD98059 (Tocris) was applied to cells at 20uM for

15 minutes prior to ligand stimulation, similar to a previously described protocol [323].

7.4.1 Immunoblot

HEK 293 cells were transiently transfected CXCR3A or CXCR3B using

Lipofectamine 2000. After 48 hours, cells were starved in serum-free DMEM >4 hours and treated with the indicated ligand for the indicated duration. Cells were lysed in ice- cold RIPA buffer containing phosphatase and protease inhibitors (Phos-STOP (Roche), cOmplete EDTA free (Sigma)) for 15 minutes, sonicated, and cleared of insoluble debris by centrifugation at 12,000 x g (4 °C, 15 min) after which the supernatant was collected.

Protein was resolved on SDS-10% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted at with the indicated primary antibody overnight (4

°C). phospho-ERK (Cell Signaling Technology, #9106) and total ERK (Millipore #06-182) were used to assess ERK activation. GRK knockdown was assessed by immunoblot of

GRK2 (Santa Cruz #sc-13143), GRK3 (Santa Cruz #sc-365197), GRK5 (Santa Cruz #sc-

11396), GRK6 (Santa Cruz #sc-377494) and protein loading assessed with alpha-tubulin

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(Sigma #T6074). Beta-arrestin levels were assessed using anti-beta-arrestin-1 (A1-CT) and anti-beta-arrestin-2 (A2-CT) antibodies kindly provided by Dr. Robert J. Lefkowitz

(Duke University) and previously validated [123]. Horseradish peroxidase-conjugated polyclonal mouse anti-rabbit-IgG or anti-mouse-IgG were used as secondary antibodies.

Immune complexes on nitrocellulose membrane were imaged by SuperSignal enhanced chemiluminescent substrate (Thermo Fisher). For quantification, phospho-ERK 1/2 relative intensity was normalized to total ERK 1/2 relative intensity using ImageLab

(Bio-Rad).

7.4.2 Intact Cell Phosphorylation

These assays were performed as described previously [324, 325]. HEK293 cells were transfected with pcDNA3.1 plasmids encoding N-terminal FLAG-tagged constructs of CXCR3A, CXCR3B, or no protein. Confluent cells in 6-well dishes were aliquoted to metabolic labeling or to cell surface immunofluorescence and flow cytometry. For metabolic labeling, cells were serum-starved overnight, rinsed and then labeled with 32Pi (100 µCi/ml, 37 °C, 1 hr) in phosphate-free Dulbecco's modified Eagle's medium, 20 mM HEPES, pH 7.4, with 0.1% (w/v) fatty acid-free bovine serum albumin

(Sigma), 100 µg/ml streptomycin and 100 units/ml penicillin. Cells were then exposed to the same medium lacking or containing 100nM CXCL11 for 5 min (37 °C), and transferred to ice. After 2 washes with Dulbecco’s PBS, cells were solubilized in “M2 buffer”: 1% (w/v) Triton X-100™, 0.05% SDS, 5 mM EDTA, 50 mM Tris-Cl, pH 8.0 (25

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°C), 200 mM NaCl, 50 mM NaF, 10 mM disodium pyrophosphate, and protease inhibitors (1 mM benzamidine, 5 ug/ml aprotinin, 10 µg/ml leupeptin). After solubilization (4 °C, 1 hr), cell lysates were cleared of insoluble debris by two sequential centrifugations at 10,000 × g (4 °C, 30 min). Supernatant aliquots were subjected to immunoprecipitation and modified Lowry protein assay (DC Protein Assay Kit, Bio-

Rad). The FLAG-CXCR3A and –CXCR3B were immunoprecipitated from 500 µl of cell lysate by inversion mixing for 60 min (4 °C) with 10 µl of agarose beads conjugated to

M2 monoclonal anti-FLAG IgG1 (Sigma-Aldrich) (these beads were suspended in M2 buffer supplemented with 3% (w/v) bovine serum albumin). Beads were then washed thrice with M2 buffer and finally incubated in 1× Laemmli buffer at 37 °C for 2 h to dissociate immune complexes, which were resolved on SDS-10% polyacrylamide gels.

Proteins were transferred to nitrocellulose membranes and subsequently processed for autoradiography with an intensifying screen at -80 °C for 2-4 days. After autoradiography, each nitrocellulose membrane was immunoblotted for CXCR3 with mouse IgG (R&D systems clone #49801), as described [326]; chemiluminescence was imaged and quantitated with a Bio-Rad ChemiDoc™ XRS+, which was also used to photograph and quantitate autoradiography films. 32P signals in each CXCR3 band were normalized to cognate receptor immunoblot signals, after subtracting nonspecific signals obtained from lanes loaded with immunoprecipitations of mock-transfected-cell lysates.

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7.4.3 Generation of beta-arrestin1/2-double knockout (∆ARRB1/2) HEK293 cells.

∆ARRB1/2 HEK293 cells were generated by Dr. Asuka Inoue through simultaneously targeting the ARRB1 and the ARRB2 genes with a CRISPR/Cas9 genome editing technology using a similar strategy that was employed for Gq/11-double knockout cells and as previously described [327, 328]. Cells were transfected with 0.8 ug of CXCR3A or 1 ug of CXCR3B with Fugene 6 (Promega) per manufacturer specifications. For beta-arrestin rescue studies, either 1 ug of beta-arrestin-1 and 1 ug beta-arrestin-2 or 2 ug empty vector (pcDNA3.1) was also transfected. Lysates were collected and immunobloted as described above.

7.4.4 Flow Cytometry to assess receptor surface expression

HEK 293 cells were transiently transfected with either CXCR3A or CXCR3B as described above. After 48 hours, transfected cells and untransfected controls were harvested with trypsin and washed twice with ice cold PBS supplemented with 1% bovine serum albumin (w/v). Cells were then resuspended in flow cytometry buffer

(PBS supplemented with 3% FBS and 10mM EDTA). One million cells were transferred to each flow cytometry tube, spun down, and blocked with flow cytometry buffer supplemented with 5% normal human serum and 5% normal rat serum for 15 minutes at 4°C. Anti-human CXCR3 phycoerythrin (PE) conjugated antibody (R&D systems clone #49801) was applied to cells at a 1:150 dilution (100 uL final volume). Cells were

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incubated at 37°C for 30 minutes and then washed with 2mL of FACS buffer, fixed for 10 minutes with 0.4% paraformaldehyde, and resuspended in FACS buffer.

7.5.1 Study subjects and skin samples

Skin biopsies and venipuncture were obtained from male and female volunteers under a protocol approved by the Institutional Review Board of Duke University.

Patches containing test allergens were applied to study participants on day 1, removed on day 3, and read at ninety-six to 120 hours. If a study participant had a positive patch test, then a 4 millimeter punch biopsy at the positive test site and a 4 millimeter punch biopsy at a negative site (normal skin) were obtained from normal regions of skin nearby. Immunofluorescence was conducted as previously described [329]. Preceding cell counting, skin images from allergen patch-tested skin and matched non-lesional

(NL) controls from the same patient were separated by color channel. All collected images were processed and analyzed in ImageJ by the laboratory of Dr. Amanda

MacLeod. The ImageJ cell counter tool recorded computer mouse clicks on cells that were labeled with colored dots. Cell numbers were expressed as counts per view.

7.5.2 Human subjects

All studies involving human subjects were approved by the Institutional Review

Board of Duke University Health System. Study participation inclusion was offered to patients undergoing patch testing in a specialty contact dermatitis clinic. Inclusion criteria were >18 years of age and completion of patch testing. Exclusion criteria were

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pregnancy, topical corticosteroids at patch site, oral corticosteroids, systemic immunosuppressants, phototherapy, known bleeding disorders and allergy to lidocaine or epinephrine.

7.5.3 Mice

Wild-type C57BL/6; C57BL/6 CXCR3−/0, CXCR3−/−; and C57BL/6 ARRB2 −/− mice were bred and maintained under specific-pathogen-free conditions in accredited animal facilities at the Duke University. C57BL/6 CXCR3−/0, CXCR3−/− mice were acquired from the Jackson Laboratory (Maine, USA, stock # 005796) and C57BL/6 ARRB2

−/− were kindly provided by Robert J. Lefkowitz. Mice were between six and fifteen weeks of age when first used.

7.6.1 Mouse allergic contact hypersensitivity (CHS)

Mice were sensitized via topical application of 0.5% DNFB (Sigma-Aldrich, St.

Louis, MO) in 4:1 acetone/olive oil on their shaved back (50 ul) and were challenged on their ears four days later with 10 ul of 0.3% DNFB or vehicle control. Then, four, twenty- four, and forty-eight hours later, 10 ul of either vehicle, VUF10661 (50 uM), or VUF11418

(50 uM) dissolved in 72:18:10 acetone:olive oil:DMSO was topically applied to the indicated ear by an investigator blinded to treatment. Ear thickness was measured at different time points using an engineer’s micrometer (Standard Gage).

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7.6.2 Chemotaxis transwell assay

Chemotaxis assays were conducted similarly to that previously described [316].

Briefly, mouse leukocytes, obtained by passing cells isolated from the spleen and subjected to erythrocyte lysis through a 70 um filter, were suspended in RPMI 1640 medium containing 5% FBS. For the assay, 106 cells in 100 ul medium were added to the top chamber of a 6.5 mm diameter, 5 um pore polycarbonate Transwell insert (Costar,

Cambridge, MA) and incubated in duplicate with the indicated concentrations of ligand suspended in 600 ul of the medium in the bottom chamber for two hours at 37°C. Cells migrating to the bottom chamber were resuspended, washed, stained with a Live/Dead marker (Aqua Dead, ThermoFisher) and antibodies to cell surface markers (CD3, CD4,

CD8, CD44, and CD45), fixed with paraformaldehyde, and subjected to cell counting flow cytometric analysis with a BD LSRII Flow cytometer. Flow cytometry was performed in the Duke Human Vaccine Institute Research Flow Cytometry Facility

(Durham, NC). We were unable to identify a reliable anti-murine CXCR3 antibody for flow cytometry suitable for surface staining. CountBright beads (ThermoFisher) were added immediately after bottom chamber resuspension to correct for differences in final volume and any sample loss during wash steps. A 1:10 dilution of input cells was similarly analyzed. Migration was calculated by dividing migrated cells by input cells.

In some studies, cells were pre-incubated with either 100 ng/mL pertussis toxin (List

Biological Laboratories), 100 uM of the PI3K inhibitor LY 294002 (Sigma-Aldrich), or 100

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nM of the Akt inhibitor AZD 5363 (Axon Med Chem) for one hour at 37°C. Human peripheral blood leukocytes were obtained by venipuncture in accordance with the

Duke Institutional Review Board, subjected to erythrocyte lysis, and assayed as above, with the addition of an anti-human CXCR3 antibody. For some samples, reliable anti- human CXCR3 staining was not obtained, which resulted in two fewer replicates in the

CD8+CXCR3+ group compared to the CD8+CD44+ group. Analysis was conducted with

FlowJo (Ashland, OR) Version 10.

7.6.3 Skin assessment of T cells

Mice were subjected to the mouse allergic contact hypersensitivity assay as described above, with the exception that both ears received 0.3% DNFB elicitation to induce inflammation. Two ears were pooled from a single mouse to produce one biological replicate. Approximately four hours after the last topical drug treatment, mouse ears were dissected and transferred dorsal side up to a dish containing ice-cold

PBS. Dorsal and ventral layers were separated and added to 5 mL of digestion media consisting of HBSS supplemented with 5% FBS (Corning), 10 mM HEPES, 0.04 mg/ml liberase (Sigma-Aldrich), and 0.3 mg/ml DNAse (Sigma-Aldrich). Ears were incubated at

37°C for ten minutes, minced, and incubated for an additional thirty minutes at 37°C, vortexing gently every ten minutes. After forty minutes, 25 mL of PBS was added, the sample was vortexed, and the sample strained through 70 um mesh into a fresh 50 mL conical tube. Cells were spun down at 4°C 200 x g, and resuspended in PBS

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supplemented with 10mM HEPES, 5mM EDTA, and 1% BSA. To obtain leukocyte cell counts, cells were manually counted by an investigator blinded to treatment group using Turks solution. Cells were transferred to round bottom FACS tubes, washed twice with 2 mL ice cold PBS, and stained with a Live/Dead marker (Aqua Dead, Thermo

Fisher # L34957). Cells were blocked in PBS supplemented with 3% FBS and 10 mM

EDTA (FACS buffer) with anti-CD16/32 (Fcγ-block), 5% normal mouse serum (Thermo

Fisher), and 5% normal rat serum (Thermo Fisher). Cells were then stained with antibodies to cell surface markers (CD3, CD4, CD8, CD44, and CD45) for thirty minutes at 4°C, washed with FACS buffer, and fixed with 0.4% paraformaldehyde. Foxp3 intracellular staining was conducted with an anti-Foxp3 antibody using a Foxp3 /

Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer protocol. Flow cytometry was performed in the Duke Human Vaccine Institute Research

Flow Cytometry Facility on a BD LSRII Flow cytometer (Durham, NC). Analysis was conducted with FlowJo (Ashland, OR) Version 10. Total cells were calculated by multiplying the relative abundance of total live cells by the manual leukocyte count.

7.6.4 Immunofluorescence of human skin

Human skin immunofluorescence experiments were conducted in the laboratory of Dr. Amanda MacLeod. Patient samples were acquired under the direct clinical supervision of Dr. Amber Atwater. Sections of frozen human specimens were incubated overnight at 4°C with primary antibodies anti-human CD3 (OKT3), Biolegend, San

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Diego, CA ); anti-human beta-arrestin (kindly provided by Robert J. Lefkowitz) and followed by reaction with Cy3 and Alexa Fluor 647-conjugated secondary antibodies

(Thermo Fisher Scientific, Waltham, MA). Next, the sections were then incubated with

Alexa 488-conjugated mouse IgG1 antibodies against human CXCR3 (R&D Systems,

Inc., Minneapolis, MN) or mouse IgG1 Isotype Control (Tonbo Biosciences, San Diego,

CA). Nuclei were counterstained with Hoechst 33342, washed in PBS, and mounted with

Anti-fade Mounting Media (Thermo Fisher Scientific, Waltham, MA).

7.6.5 Targeted phosphoprotein analysis

Targeted phosphoprotein analyses were conducted at PrimityBio (Fremont, CA) under the direction of Dr. Thomas Wehrman. Briefly, human peripheral blood mononuclear cells were stimulated with saturating concentrations of the indicated ligands for one, two, five, or fifteen minutes, fixed with 1% PFA for ten minutes at room temperature, and were prepared for antibody staining as previously described [330].

Briefly, the fixed cells were permeabilized in 90% methanol for fifteen minutes. The cells were then stained with a panel of antibodies specific to the markers indicated (Primity

Bio Pathway Phenotyping service) and analyzed on an LSRII flow cytometer (BD

Biosciences). The log2 ratio of the mean fluorescence intensity of the stimulated samples divided by the unstimulated control samples were calculated as a measure of response.

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7.7 Statistical Analyses

Dose-response curves were fitted to a log agonist vs stimulus with three parameters (Span, Baseline, and EC50) with the minimum baseline-corrected to zero using Prism 7.0 (GraphPad, San Diego, CA). For comparing ligands in concentration- response assays, a two-way ANOVA of ligand and concentration with all four ligands was first conducted. If a significant ligand by concentration interaction was observed

(p<0.05), then comparative two way ANOVAs between individual ligands were conducted and corrected for multiple comparisons (e.g., for studies with four ligands, p

< 0.05/6 was used as the cutoff for statistical significance), with comparisons finding a significant effect of ligand noted with the figures. Further details of statistical analysis and replicates are included in figure captions. Error bars shown in dose response analysis signify the standard error of the mean unless otherwise noted. * indicates p <

0.05 throughout this dissertation to indicate statistical significance from pertinent comparisons detailed in the figure legends, unless otherwise noted.

8. Conclusions

In this work, we demonstrate that endogenous biased signaling between G protein and beta-arrestin pathways exists at CXCR3 with its endogenous ligands

CXCL9, CXCL10, and CXCL11. We also demonstrate that modifications of the CXCR3 to the extracellular N-terminus can alter intracellular coupling, as CXCR3-B, a splice

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variant with an extended N-terminus, behaved as a beta-arrestin biased receptor relative to CXCR3-A.

Here, we identify that CXCL11 is beta-arrestin-biased chemokine relative to

CXCL9 and CXCL10. We also identify small molecule biased agonists for CXCR3, which serve as useful tools for dissecting the physiological relevance of biased signaling in chemotaxis and inflammatory models.

With our findings, it is unsurprising that CXCR3 small molecule screens that focus primarily on affinity or G protein signaling could potentially underperform in identifying promising preclinical agonists or antagonists. Indeed, the role of beta- arrestin in other CKR signaling pathways suggests that an expanded screen to include the beta-arrestin pathway (and potentially Akt) would improve candidate selection. In summary, targeting different CXCR3 pathways with biased agonists with similar affinity but different efficacy for G protein and beta-arrestin pathways produced distinct physiological differences in chemotaxis and contact hypersensitivity reactions, which have strong implications for drug development within the chemokine receptor family.

Future experiments defining how bias is encoded at the receptor and the development of tools to quantify GPCR signaling complex components would aid in drug design and screening.

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Biography

Jeffrey S Smith was born on September 17, 1987 to Elaine G Chapman and

Richard D Smith in Richland, WA. Raised in Richland, WA he attended Hanford High

School, graduating as valedictorian. Jeffrey then attended the University of Washington from 2006-2010, where as a member of the University Honors Program he earned a

Bachelor of Science degree in Neurobiology and minored in Chemistry, graduating magna cum laude. He then worked as a research technician at the University of

Washington from 2010-2012 in the laboratory of Dr. Charles Chavkin, studying opioid receptor signaling. Jeffrey subsequently chose to matriculate in Duke University’s

Medical Scientist Training Program in 2012 to continue to study receptor signaling. In the Duke School of Medicine, he was inducted into the Alpha Omega Alpha Honor

Medical Society. Jeffrey joined the Duke Department of Biochemistry in 2014, where he was mentored primarily by Dr. Sudarshan Rajagopal, MD/PhD and co-mentored by Dr.

Robert J. Lefkowitz, MD. During his thesis work, his publications include first author articles in the Journal of Biological Chemistry, Molecular Pharmacology, and Nature Reviews

Drug Discovery, with additional manuscripts under review.

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