Development and Validation of a Novel Quantitative Assay for Cell

Development and Validation of a Novel Quantitative Assay for Cell Surface expression of GPCRs using a Receptor β-lactamase Fusion Protein and the Colourometric Substrate Nitrocefin

Vincent Lam

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Pharmacology and Toxicology University of Toronto

Development and Validation of a Novel Quantitative Assay for Cell Surface Expression of GPCRs using a Receptor β-lactamase Fusion Protein and the Colourometric Substrate Nitrocefin

Vincent Lam

Master of Science

Department of Pharmacology and Toxicology University of Toronto

2013 Abstract

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of

GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

Acknowledgments

I would like to thank the following people who have made completion of the thesis possible.

 First I would like to express my thanks and gratitude to Dr. Ali Salahpour for his guidance, mentorship, support, and endless positive encouragement throughout the completion of this thesis.

 I would also like to thank Dr. Jane Mitchell (co-supervisor) for her advice and critical analyses of my work.

 I would also like to thank Dr. Amy Ramsey and Dr. David Riddick (Advisor) for their support, guidance, and suggestions.

 Furthermore I would like to thank the members of the Ramsey and Salahpour lab for their support and intellectual discussion during my studies.

 Lastly I would like to thank my friends and family for their endless encouragement and support throughout my time in graduate studies, without them this thesis would not have been possible.

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Table of Contents

Acknowledgments ...... iii Table of Contents ...... iv List of Tables ...... vi List of Figures ...... vii List of Abbreviations ...... viii List of Appendices ...... x

Chapter 1 Introduction ...... 1 Specific Aims and Working Hypothesis ...... 1 1.0 GPCR Pharmacology ...... 2 1.1 GPCR Classification ...... 3 1.2 Structure and function ...... 4 1.3 GPCR Signalling ...... 5 1.3.1 Ligand binding to receptor ...... 5 1.3.2 G-proteins ...... 7 1.3.3 Effectors ...... 8 1.4 GPCR Trafficking from the ER ...... 9 1.4.1 Signal Sequences ...... 10 1.4.2 Post translational modifications ...... 12 1.4.3 Molecular Chaperones ...... 14 1.4.4 Pharmacological Chaperones and Diseases ...... 16 1.5 GPCR Oligomerization ...... 17 1.6 GPCR Endocytic Trafficking ...... 18 1.6.1 G-protein Coupled Receptor Kinases (GRK) ...... 19 1.6.2 Arrestins ...... 20 1.6.3 Tonic/Constitutive Internalization ...... 22 1.6.4 Endocytic pathway ...... 22 1.7 Summary of Assays for measuring surface expression of GPCRs ...... 23 1.7.1 Fluorogen Activating Protein Biosensor ...... 25 1.7.2 Internalization Assays ...... 26 1.7.3 N-terminal GPCR Fusion Tags ...... 27 1.8 β-lactamase Assay ...... 28

Chapter 2 Materials and Methods ...... 33 2.1 Reagents ...... 33 2.2 Plasmid Construction ...... 33 2.3 Cell Culture ...... 33 2.4 Generation of Stable Cell Lines and Transient Transfections ...... 34 2.5 Western Blotting ...... 34 2.6 βlac-β2AR Immunofluorescence ...... 34 2.7 βlac-β2AR Functional Assay using BRET EPAC cAMP Biosensor ...... 35 2.8 βlac Assay ...... 35 2.9 ELISA ...... 35 2.10 βlac-β2AR Agonist Studies...... 36 2.11 βlac-β2AR Antagonist Studies ...... 36

2.12 βlac-β2AR Z’ Determination for Agonist Induced Internalization...... 36 2.13 GBR1 Molecular Chaperoning Studies ...... 36 2.14 Data Analyses ...... 37

Chapter 3 Results ...... 38 3.1 Generation of the βlac Plasmids and Stable Cell Lines Expressing βlac-GPCR Fusion Constructs ...... 38 3.2 Trafficking and Signalling of the βlac-β2AR ...... 38 3.3 βlac-β2AR experiments ...... 42 3.3.1 Comparison of Isoproterenol stimulated β2AR Internalization using the βlac and the ELISA Assays ...... 42 3.3.2 Antagonist Blocking of Isoproterenol Induced Internalization of the β2AR ...... 46 3.3.3 Z’ Determination of the SS-HA-βlac-β2AR Internalization ...... 46 3.3.4 Pharmacological Chaperoning using β2AR Antagonists ...... 48 3.4 Comparison of GBR1 Surface Expression using the βlac and the ELISA ...... 50

Chapter 4 Discussion ...... 52 4.1 Summary of Key Findings ...... 52 4.2 Nitrocefin Permeability ...... 52 4.3 Functional Experiments with the SS-HA-βlac-β2AR ...... 52 4.4 SS-HA-βlac-β2AR Internalization ...... 55 4.5 Pharmacological chaperoning ...... 57 4.6 Z’ Factor of βlac-β2AR internalization ...... 58 4.7 SS-HA- βlac-GBR1 Surface Expression by Co-expression with GBR2 ...... 60 4.8 Cost Analyses ...... 60 Conclusion ...... 61 References ...... 63 Appendices ...... 83

List of Tables

Table 1: Comparison of time and cost of ELISA and βlac assays ...... 62

List of Figures

Figure 1.1: The conventional GPCR activation model of GPCR signaling ...... 6

Figure 1.2: Schematic of the βlac assay ...... 29

Figure 1.3: Nitrocefin as a chromogenic substrate for the βlac assay ...... 30

Figure 1.4: Mechanism of action for a class A β-lactamase ...... 32

Figure 3.1: Western blot of stable clonal cell line expression levels of SS-HA-βlac-GBR1 and SS-HA-βlac-β2AR ...... 39

Figure 3.2: Immunofluorescence of HEK293 cells stably expressing SS-HA-βlac-β2AR ...... 41

Figure 3.3: β2AR functional assay using the BRET cAMP EPAC biosensor ...... 43

Figure 3.4: Comparison of βlac and ELISA with the SS-HA-βlac-β2AR stable cell line ...... 44

Figure 3.5: Blocking of isoproterenol induced internalization with pre treatment of antagonists 45

Figure 3.6: Z’ of the βlac assay using the SS-HA-βlac-β2AR stimulated with isoproterenol ...... 47

Figure 3.7: Overnight incubation with alprenolol and propranolol increases surface expression of SS-HA-βlac-β2AR ...... 49

Figure 3.8: Comparison of βlac assay and ELISA Surface expression of GBR1 with GBR2 co- expression ...... 51

Figure 4.1: Representative diagram for the BRET EPAC cAMP Biosensor ...... 54

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List of Abbreviations

A1AR α1 adrenergic receptor ANOVA Analysis of variance AP2 Adaptor protein 2

β1AR β1 adrenergic receptor

β2AR β2 adrenergic receptor

β3AR β3 adrenergic receptor βlac β-lactamase cAMP Cyclic adenosine monophosphate COPII Coat protein 2 CRLR Calcitonin receptor-like receptor C-terminal Carboxyl-terminal DAG Diacylglycerol DOR δ-opioid receptor DRIP 78 Dopamine receptor-interacting protein

EC50 Median effective concentration at 50% ELISA Enzyme linked Immunosorbent Assay EPAC Exchange proteins activated by cAMP ER Endoplasmic Reticulum FAP Fluorogen activating protein GABA γ-aminobutyric acid GBR1, GBR2 Metabotropic GABA bR1, bR2 GDP Guanosine diphosphate GFP Green fluorescent protein GRK G-protein coupled receptor kinase GPCR G-protein coupled receptor GTP Guanosine triphosphate HA Hemaglutinin HEK 293 Human embryonic kidney cells HRP Horseradish peroxidase viii

HTS High throughput screening

IC50 Median inhibitory concentration at 50%

IP3 Inositol 1,4,5-trisphosphate mGluR Metabotropic glutamate receptor MRAP Melanocortin-2 receptor accessory protein NDI Nephrogenic diabetes insipidus N-terminal Amino-terminal PAR1 Protease activated receptor 1

PIP2 Phosphatidylinositol 4,5-bisphosphate PKA, PKC Protein kinase A, C PLCβ Phospholipase C β RAMP Receptor activity modifying proteins REEP Receptor expression enhancing protein RTP Receptor transporting protein RGS Regulators of G protein signalling Rluc Renilla luciferase TAAR1 Trace amine associated receptor 1 TM Transmembrane TPβ Thromboxane A2 β receptor V1R Vasopressin 1 receptor V2R Vasopressin 2 receptor WT Wild type YFP Yellow fluorescent protein

List of Appendices

Appendix Figure 1: Cell permeability of the βlac substrate nitrocefin ...... 83

Appendix Figure 2: Dose response of isoproterenol mediated internalization quantified with flow cytometry ...... 84

Appendix Figure 3: Time course of isoproterenol mediated internalization quantified with flow cytometry ...... 85

Appendix Figure 4: Functionality of SS-HA-βlac-GBR1 using the BRET EPAC cAMP biosensor ...... 86

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Chapter 1 Introduction

Specific Aims and Working Hypothesis

Surface expression of G-protein coupled receptors (GPCR) is crucial for the correct function of the receptor. Within the past 20 years proper trafficking of GPCRs and other membrane proteins has seen an increase in interest within the scientific community. Specifically the trafficking of GPCRs to the plasma membrane is of interest. Indeed there are an increasing number of GPCRs that require the use of specific molecular chaperones to aid in their proper trafficking to the plasma membrane (see section 1.4.3). In addition there are an increasing number of diseases that are linked to alterations in normal trafficking of GPCRs and other membrane proteins (see section 1.4.4). Therefore there is a need for fast and robust assays that can quantify surface expression and the effects of small molecules or proteins on the trafficking of GPCRs. Unfortunately the current assays that are commonly used for measuring surface expression of GPCRs, while being robust and quantitative, are time consuming and relatively low throughput. In this context, an assay that is capable of high throughput screening would provide a powerful tool in the search for specific ligands or proteins that can affect the surface expression of membrane proteins. Indeed, within the last 5 years, there has been several novel assays created that address the current limitations as discussed above (see section 1.8). Here we describe a new assay for the quantification of GPCRs on the plasma membrane using an N- terminal β-lactamase-GPCR fusion protein (βlac-GPCR). We hypothesize that this new assay has the ability to quantify receptor surface expression in an equivalent manner to current standard assays such as the enzyme linked immunosorbent assay (ELISA). In addition we also aim to optimize and miniaturize this assay for use in a high throughput manner. We validated this assay according to the following aims with well-established trafficking profiles of two separate GPCRs.

Aim 1: β2-adrenergic receptor (β2AR) internalization

The β2-adrenergic receptor (β2AR) is a prototypical GPCR that has been used to validate multiple surface expression assays (Hammer et al., 2007; Fisher et al., 2010; Yano et al., 2012). In our first aim, we created a stable cell line expressing a βlac-β2AR construct which was used to

validate our assay. The internalization of β2AR is well characterized with reproducible EC50 and half-life values for internalization upon stimulation with isoproterenol, a β2AR full agonist. Taking advantage of this internalization profile, we compared the βlac assay in parallel with an ELISA. In addition, we looked at the ability of antagonists to block ligand induced internalization of β2AR using the βlac assay. Lastly it has been shown that the β2AR/β1AR selective antagonists alprenolol and propranolol can act as pharmacological chaperones increasing the surface expression of the β1AR and potentially β2AR (Kobayashi et al., 2009). We used the βlac assay in order to assess whether these antagonists can increase surface expression of the β2AR. In summary we compared the ability of our new βlac assay with an ELISA for the general internalization profile of β2AR. This profile includes the time course and dose response of isoproterenol mediated internalization as well as antagonist blockade of isoproterenol induced internalization. Lastly, pharmacological chaperoning effects of alprenolol and propranolol were assessed by overnight treatment of βlac-β2AR cells.

Aim 2: GΑBA b R1 (GBR1) surface expression

In the second aim we used the metabotropic GΑBA b R1 (GBR1) receptor. The GBR1 receptor has a well characterized surface expression profile where the receptor is retained in the endoplasmic reticulum (ER) unless co-expressed with its molecular chaperone GΑBA bR2 (GBR2). We compared the ability of the βlac assay and ELISA to measure surface expression of GBR1 by transiently transfecting GBR2 into stable cell lines expressing a βlac-GBR1.

Overall, we validated the βlac assay as being quantitatively equivalent to an ELISA while being less expensive, more robust and higher throughput than an ELISA.

1.0 GPCR Pharmacology

With over 800 known members, GPCRs are the largest family of receptors in the human genome (Audet and Bouvier, 2012) where these receptors are currently the target of approximately 40% of the prescribed drugs on the market (Wise et al., 2002). These receptors play pivotal roles in a wide range of biological functions that include but are not limited to: olfaction, cardiovascular function, and neurotransmission (Ferguson et al., 1998). In the introduction, we will present a general overview on the current state of GPCR knowledge with specific emphasis on GPCR trafficking and assays for measuring surface expression.

1.1 GPCR Classification

With over 800 known receptors, GPCRs represent the largest class of receptors in the human genome (Foord, 2002). Although GPCRs all share similar signalling mechanisms and receptor topology, there is very little sequence homology shared between receptors. Therefore GPCRs have been divided into five or six (depending on classification criteria) major families of receptors based primarily on sequence similarity (Foord et al., 2005). The vast majority of GPCRs are classified into the three following classes: Class A (rhodopsin-like), B (secretin-like), and C (metabotropic glutamate-like) (Foord et al., 2005). The details regarding the classification criteria for GPCRs are beyond the scope of this study, for a detailed description please see the following review from the International Union of Pharmacology (Foord, 2002).

Class A GPCRs (rhodopsin-like) comprise the largest family of GPCRs with roughly 700 genes found in the human genome (Fredriksson et al., 2003). It is important to note that olfactory receptors make up the bulk of the class A family of receptors consisting of over 65% of the receptors within this class. Class A receptors in general have two conserved motifs. The first motif is the DRY motif found in the third transmembrane domain; where this motif has been shown to be involved in the activation of various members of the class A family through an ‘ionic lock’ with the sixth transmembrane domain (Scheer et al., 1996; Rasmussen et al., 1999; Ballesteros et al., 2001; Rovati et al., 2007). In addition the NPXXY motif is also a highly conserved motif within this family of receptors and is involved in signalling as well as internalization of GPCRs (Bouley et al., 2003; Fredriksson et al., 2003).

Class B GPCRs (secretin-like) are characterized by their large (~100 amino acid) cysteine rich N-terminal domain responsible for ligand binding. In contrast to class A receptors, class B receptors bind peptides as their endogenous ligands (Harmar, 2001). Further distinction between Class B and Class A receptors are the absence of the DRY and NPXXY motifs in class B receptors. However it has been proposed that the motifs REY and VAVLY act as equivalent motifs to the DRY and NPXXY motifs respectively for class B receptors (Frimurer and Bywater, 1999; Vohra et al., 2013).

Class C receptors (metabotropic-glutamate like) are characterized by their large 500-600 amino acid N-terminal domains. The large N-terminal domain binds the ligands and consists of a two lobed structure termed the ‘venus fly trap’ domain for the mechanism by which it binds

4 ligands (Bräuner-Osborne et al., 2007). However, the mechanisms by which the binding of a ligand to the venus fly trap domain translates into GPCR signalling remains elusive (El Moustaine et al., 2012). In addition, a unique distinction for class C receptors is the finding that all these receptors function as dimers (Bräuner-Osborne et al., 2007; El Moustaine et al., 2012). Interestingly a subset of class C receptors contain the DRY motif found in class A receptors (Pin et al., 2003). Lastly class C receptors contain a well conserved xPKxY domain that has been proposed to function similarly to the NPXXY domain of class A receptors (Pin et al., 2003).

1.2 Structure and function

GPCRs are 7-transmembrane integral proteins localized predominately at the plasma membrane with evidence of some receptors localized to intracellular membranes as well (Tadevosyan et al., 2012). All GPCRs share the same general topography: a plasma membrane spanning 7-transmembrane hydrophobic core, three intracellular loops, three extracellular loops, an extracellular N-terminal domain, and an intracellular C-terminal domain (Baldwin, 1993; Bockaert and Pin, 1999; Duvernay et al., 2005). Although this topology and organization of GPCRs has been proposed for many years, this layout has only been recently confirmed with the crystallization of mammalian GPCRs starting with bovine rhodopsin (Palczewski et al., 2000). Due to a methodological breakthrough there has recently been a large increase of solved crystal structures starting with the 2AR (Rasmussen et al., 2007). Indeed at the time of writing of this section, there are 57 structures solved for 13 distinct GPCRs all within the class A family of GPCRs (see section 1.1). These crystal structures have confirmed the structure of GPCRs illustrating the high diversity of orthosteric binding sites, as well as revealing the crucial link between ligand binding and receptor activation. Even though early structures were crystallized with agonists or antagonists bound, a surprising observation has been the similarity of the tertiary structure for these ligand bound GPCRs (see review Audet and Bouvier, 2012). Indeed the apparent similarity between antagonist and agonist bound structures can be attributed to the lack of co-crystallization of the G-proteins, validating the model that GPCR activation requires both the binding of ligand and the presence of the G-protein.

Fortunately, recent innovation in the crystallization methods have also yielded receptors co crystallized with G-proteins where further insight into GPCR activation has been achieved through the analysis of three separate crystal structures. The first was the crystallization of the

5 ligand free form of opsin (active-like state of rhodopsin) with co-crystallization of the C-terminal domain of transducin, the Gα subunit for rhodopsin (Scheerer et al., 2008). This structure when compared to the inactive rhodopsin structure, showed an outward movement of TM VI which forms the binding site for transducin. Subsequently two additional structures added to this observation and confirmed the movement of TM VI as a function of G-protein binding and activation. These structures were the co-crystallization of the agonist bound β2AR and the heterotrimeric G-protein (Rasmussen et al., 2011a) and the agonist bound β2AR with a nanobody mimicking Gαs (Rasmussen et al., 2011b). Additionally, these structures provided a mechanism for how GPCRs facilitate nucleotide exchange upon the activation of the receptor. In the activated agonist bound GPCR, the outward movement of the TM VI domain causes a 130° rotation of the Gα subunit. It is proposed that this rotation in the Gα subunit allows for the exchange of GDP for GTP (Rasmussen et al., 2011b). Although these structures have provided invaluable information for how GPCRs are activated, there are still some questions that remain. For instance, this model shows one receptor activating one G-protein complex, however it is well established that some functional receptor complexes require GPCR dimers (see section 1.5). Therefore it is still unclear how these dimers affect signalling or binding to G-proteins.

1.3 GPCR Signalling

The simplest model of GPCR signalling involves three steps. First, ligand binding to the receptor, where each family of GPCR, in general, has specific endogenous ligands that bind the receptors with high affinity. Once an agonist is bound and the receptor is activated the second step of GPCR signalling occurs, whereby the receptor acts to catalyze the exchange of GDP for GTP on the Gα subunit of the heterotrimeric Gαβγ G-protein complex. The third step involves the Gα subunit and Gβγ dimer interacting with membrane bound/associated effector proteins or ion channels leading to an increase/decrease in cytosolic secondary messengers (see Figure 1.1 for more detail of this model) (Luttrell, 2006).

1.3.1 Ligand binding to receptor

The first step of GPCR signalling involves the binding of the extracellular ligand to the GPCR. GPCR ligands are separated into two separate classes: agonists and antagonists. Agonists are ligands that promote receptor activation while antagonists are ligands that block activation of the receptor. Antagonists can be further separated into two subclasses: inverse-agonists, ligands

Figure 1.1: The conventional GPCR activation model of GPCR signaling. There are three main components to this model of signaling. First an extracellular hormone/ligand (H) binds to the 7-TM protein that is coupled to a heterotrimeric G-protein. Upon hormone binding, the receptor is activated and catalyzes the exchange of GDP to GTP for the Gα subunit of the heterotrimeric G-protein complex. The binding of GTP causes the trimer to dissociate into the GTP-Gα subunit and the Gβy heterodimer. These dissociated G-proteins activate effector proteins located on the plasma membrane which activate or inhibit secondary messenger production. Typical effector proteins include enzymatic effectors (ie adenylyl cyclase and phospholipases) and ion channels. Signaling is halted once the intrinsic GTPase of the Gα subunit hydrolyzes the GTP to GDP (Luttrell, 2006).

7 that block intrinsic activity of a GPCR, and neutral antagonists that have no effect on the activation state of the receptor (Audet and Bouvier, 2012). In order to predict and correctly model GPCR signalling using a mathematical model, several factors have to be considered. First and most importantly, the binding of a ligand to the receptor can be affected by the presence or absence of G-proteins. This was first observed in the 2AR where it was shown that the receptor exists in high and low affinity receptor states for the binding of ligands to the receptor. To account for this observation it was discovered that G-protein binding to the receptor acts as an allosteric modulator that can alter the affinity state of the receptor for ligands. Therefore to model this effect, the ternary complex model (De Lean et al., 1980) was created to predict agonist binding and the effects of an allosteric modulator, in this case G-proteins. Furthermore the discovery of GPCR constitutive activity (receptor activity in the absence of agonist binding) led to the addition of active (R*) and inactive (R) states of the receptor in the extended ternary complex (Samama et al., 1993). Although the extended ternary complex model is the most accepted model for GPCR signalling through G-proteins, other models have been generated to take into account the effects of biased signalling (Whistler and von Zastrow, 1998; Holloway et al., 2002; Kohout et al., 2004; Swaminath et al., 2004) and arrestin binding (Gurevich et al., 1997) on effects of ligand binding to the GPCR. For a detailed description of current state of receptor ligand binding and activation, refer to the following reviews (Kenakin, 2002, 2003, 2011).

1.3.2 G-proteins

The second component for GPCR signalling is the activation and dissociation G-proteins from the receptor. The heterotrimeric G-protein complex is composed of three subunits: the Gα

GTPase, Gβ, and Gγ. The complex is associated together when the Gα subunit is in its inactive GDP bound form. In its inactive form, the heterotrimeric G-protein is associated with the receptor complex. As described in figure 1.1, GPCR activation induces the exchange of GDP to GTP in the Gα subunit causing the heterotrimer to dissociate from the receptor into membrane tethered Gα and Gβγ subunits. Once dissociated, the Gα and Gβγ subunits interact with membrane bound effector proteins. The subsequent response is determined by the structural composition of the G-protein subunits. There are 16 genes encoding for Gα subunits yielding a total of 20 expressed Gα subunits through alternative splice variants. The Gα subunits can be separated into four families based upon sequence homology: Gαs, Gαi, Gαq, and Gα12. The Gαs

8 family (Gαs and Gαolf) of G-proteins are stimulatory toward adenylyl cyclase causing an increase in intracellular cAMP. Conversely the Gαi family (Gαi1-3, Gαt1-2, Gαo1-2, Gαz and Gαgust) are in general inhibitory to adenylyl cyclase causing a decrease in intracellular cAMP, although Gαt is an activator for the cGMP phosphodiesterase 6 (Hingorani and Ho, 1987). The Gαq family (Gαq, Gα11, Gα14, and Gα15) activates membrane associated phospholipase C β

(PLCβ) forming inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2). Lastly the Gα12 family (Gα12 and Gα13) activates Rho-specific guanine nucleotide exchange factors (Tanabe et al., 2004; Luttrell, 2006). In addition to the Gα subunits the Gβγ heterodimers also have important functions in GPCR signalling. Currently, there are 5 and 12 known Gβ and Gγ subunits respectively. Although there are almost 1000 possible combinations of Gβγ heterodimers with Gα subunits, whether all these combinations exist in vivo is currently unknown (see review Hildebrandt, 1997). Nevertheless there are common combinations of the Gβγ heterodimer found expressed in specific tissues. For example the Gβ1γ1 is the predominant heterodimer found associated with the Gαt in the retina (Ford et al., 1998). Although it was not initially recognized, it is now generally accepted that Gβγ subunits can mediate as many functions as Gα subunits (Milligan and Kostenis, 2006) and activate or inhibit effectors on their own such as the PLC-β isoforms, adenylyl cyclase, ion channels, and GRKs (Ford et al., 1998).

1.3.3 Effectors

The last component of GPCR signalling are the effectors that are modulated by the binding of the G-protein subunits (Clapham and Neer, 1993). The most well studied effectors are the family of adenylyl cyclases that convert intracellular ATP to the secondary messenger cyclic adenosine monophosphate (cAMP). The 10 members of the 12-transmembrane adenylyl cyclase family of proteins can all be activated by Gαs where most adenylyl cyclases are inhibited by Gαi or Gβγ G-proteins (Sunahara et al., 1996). Lastly it has been shown that Gβγ can also interact and activate or inhibit adenylyl cyclase (Rebois et al., 2006; Wang and Burns, 2006; Boran et al., 2011). Once the adenylyl cyclase has been activated the secondary messenger, cAMP, in turn activates further downstream signalling cascades. Some important proteins that are directly activated by cAMP include, but are not limited to, the cAMP-dependent protein kinases (PKA) and cAMP-regulated guanine nucleotide exchange factors (EPAC) (Zwartkruis and Bos, 1999).

In addition to adenylyl cyclases, the second major class of effector proteins are the PLCβ family (Isoforms 1-4). These lipases hydrolyze membrane bound PIP2 to yield the intracellular second messenger IP3 and the membrane bound DAG. The cytosolic IP3 binds to ligand gated calcium channels found on the ER releasing calcium into the cytoplasm. Meanwhile DAG is the primary activator of several isoforms of the membrane bound protein kinase C (PKC) (Morris and Scarlata, 1997).

Lastly ion channels can be inhibited or activated by G-protein subunits. For example, the ‘N-type’ calcium channels are inhibited by Gαo and βγ (Albert and Robillard, 2002), the ‘L- type’ calcium channels are activated by Gαs, and the inward rectifying muscarinic gated potassium channels are regulated by the Gβγ subunits (Wickman and Clapham, 1995; Rebois et al., 2006).

The main mechanism of stopping GPCR signalling is through the hydrolysis of the GTP to GDP by the Gα subunit. This GTPase activity of the Gα subunit can be enhanced by a family of regulators of G protein signalling (RGS) proteins. For details on the role of RGS see the following review (Kach et al., 2012). Further desensitization occurs at the receptor level and is explained in section 1.6.

Although GPCR signalling through G-proteins has been well established as the canonical signalling pathway, over the last decade, extensive studies have shown that GPCRs can also signal in non-canonical pathways that are G-protein independent (see section 1.6.2).

1.4 GPCR Trafficking from the ER

Receptor trafficking is a dynamic process that contributes to the regulation and proper function of GPCRs. While the endocytic pathway has been studied extensively for GPCRs, the molecular mechanisms for GPCR trafficking from the ER to the plasma membrane is less understood (Duvernay et al., 2005; Dong et al., 2007). The general mechanism for ER export of GPCRs first involves translation and folding of the receptor in the ER, where the receptors are subsequently packaged into transport vesicles and targeted to the ER-Golgi intermediate complex. Incomplete or misfolded receptors in the ER are ubiquitinated and degraded in a process known as ER-associated degradation (Meusser et al., 2005). During receptor maturation various post translational modifications (see section 1.4.2) occur while the receptor is transported

10 from the ER to the ER-Golgi intermediate complex and finally to the trans-Golgi network before being delivered to the plasma membrane (see review Duvernay et al., 2005). For GPCRs, trafficking and transport from the ER to the plasma membrane is generally considered the rate limiting step in their biogenesis (Petaja-Repo et al., 2000). The following section will give a brief overview of the different factors that can affect the trafficking of GPCRs from the ER to the plasma membrane.

1.4.1 Signal Sequences

The first determinant for cell surface targeting is found in the primary amino acid sequence in the form of distinct and conserved motifs. It is well established that specific motifs aid in the export of membrane proteins from the ER. In general, there are two classes of ER export motifs for non-GPCRs. The first is a diacidic DXE motif (Nishimura and Balch, 1997) that is required for the proper surface expression of transmembrane proteins such as CFTR (Wang et al., 2004) and the Kir2.1 potassium channel (Ma et al., 2001). The second class is the dihydrophobic (FF) motif that is required for the trafficking of the following membrane proteins: ERGIC-53 (Nufer et al., 2003), Erv41-46 (Otte and Barlowe, 2002), and p24 family of proteins(Dominguez et al., 1998).

In contrast, GPCRs have no consensus export motifs that are found between different classes of receptors. Nevertheless there are receptor specific motifs located on the membrane proximal C-terminal domain of GPCRs that are essential for the proper ER export. To date four distinct C-terminal motifs required for ER export of specific GPCRs have been identified. These motifs include the following: E(X)3LL on the vasopressin 2 receptor (V2R) (Schülein et al.,

1998), F(X)3F(X)3F on the Dopamine D1 receptor (Bermak et al., 2001), FN(X)2LL(X)3L on the vasopressin 1B and vasopressin 3 receptors (Robert et al., 2005), and F(X)6LL on the α2 adrenergic receptor (α2AR) and angiotensin II type 1 receptor (Duvernay et al., 2004). Interestingly although these motifs are distinct they all share the common feature of having hydrophobic residues spaced apart in such a way as to reside on one side of an alpha helix. Despite sharing similar chemical features, it is important to note that these motifs are receptor specific. For instance mutating the FN(X)2LL(X)3L motif of the vasopressin 1B receptor to

F(X)6LL found on the α2AR, caused the receptors to be retained in the ER rather than being

targeted to the plasma membrane indicating that the FN(X)2LL(X)3L motif is necessary for proper surface expression of the vasopressin 1B receptor (Robert et al., 2005).

In addition to the motifs located on the C-terminus, the intracellular loops and even the N-terminus of GPCRs can also regulate export from the ER to the plasma membrane. For example, in the α2bAR a triple arginine motif in the third intracellular loop is required for proper binding of COPII transport vesicles that mediate receptor export from the ER (Dong et al., 2012). In numerous other class A GPCRs, a single conserved leucine in the intracellular loop 1 is required for the proper export of the receptors from the ER (Duvernay et al., 2009). Lastly the motif ALAAALAAAAA in the α2cAR is found on the extracellular N-terminus and functions to aid the trafficking of the receptor to the plasma membrane (Angelotti et al., 2010).

In general the interaction between the coat protein 2 (COPII) in transport vesicles and the non-GPCR-ER export motifs is the main mechanism of membrane protein export from the ER. It is well established that the DXE and FF motifs, present in non-GPCRs, interact with components of the COPII complex to mediate ER export (Miller et al., 2003). However for GPCRs, the mechanisms by which the above mentioned ER export motifs mediate surface expression is currently unknown. It is hypothesized that these motifs traffic GPCRs to the plasma membrane by one of the following mechanisms. Firstly, like the non-GPCR export motifs, the interaction of these motifs with proteins in the COPII complex has been suggested. The only evidence for direct binding of GPCRs to a COPII complex are from studies on a triple arginine motif present in the third intracellular loop of several GPCRs (Dong et al., 2012). The second potential mechanism is through the direct binding of GPCR with specific molecular chaperones. Indeed this mechanism has been shown for the F(X)3F(X)3F motif of the dopamine D1 receptor which interacts with the ER chaperone dopamine receptor-interacting protein 78 (DRIP 78) (Bermak et al., 2001). The third mechanism involves the participation of these motifs in the dimerization with other receptors. However there is no evidence to support this mechanism. For example mutation of the F(X)6LL motif in the α2bAR retains the receptors ability to form dimers (Zhou et al., 2006). Lastly it is hypothesized that the motifs themselves aid in the folding and trafficking of the receptor in a yet unknown mechanism (Duvernay et al., 2005). In the end it can be seen that ER export motifs are important for certain receptors, however the specific mechanism of how these motifs aid in ER export appears to be different for each receptor.

In contrast to ER export motifs, some receptors contain ER retention motifs on the C- terminus. As with ER export motifs, there are three conserved ER retention motifs (KDEL, KKXX, and RXR (Dong et al., 2007)) found for membrane proteins. However, only one of the motifs mentioned above has been found to be present in GPCRs (the RXR motif). The RSRR motif of the GBR1 acts to retain the receptor in the ER and only upon binding with GBR1’s molecular chaperone, GΒR2, does the retention motif become masked (Margeta-Mitrovic et al., 2000a). In addition, the metabotropic glutamate receptor splice variant 1b is retained in the ER due to the presence of the RRKK motif in the C-terminus (Chan et al., 2001). Unlike the GBR1 the mechanism by which mGluR1b is trafficked to the plasma membrane is unknown. Although ER export motifs can be present in both the C-terminus and intracellular loops, it is important to note that the ER retention motifs only retain receptors if present on the C-terminus. For example the V2R contains two RXR motifs in the third intracellular loop, however only truncation mutants of this receptor, where the motifs are now present at the C-terminus, show a phenotype of ER retention (Hermosilla and Schülein, 2001).

As noted above, the presence of specific motifs on the intracellular portion of the GPCR is important for either the export or retention in the ER. However unlike other membrane proteins, there is a lack of consensus motifs that are present in more than one receptor indicating that the vast majority of receptors are targeted to the plasma membrane via a mechanism that is currently not known.

1.4.2 Post translational modifications

GPCRs go through a number of post translational modifications that can affect function and expression of the receptors. In general, most receptors undergo N-linked glycosylation (Renthal et al., 1973) as well as O-linked glycosylation (Sadeghi and Birnbaumer, 1999; Petaja- Repo et al., 2000; Nakagawa et al., 2001). N-linked glycosylation is the most common post translational modification and occurs at the consensus sequence NXS/T (Nita-Lazar et al., 2005). N-linked glycosylation is reported to affect the targeting of GPCRs to the cell surface although this effect is receptor specific. For example the mutation of two N-terminal glycosylation sites on the β2AR show a marked decrease in the surface expression of the receptor but with no change in function for the receptor that is on the plasma membrane (Rands et al., 1990). Furthermore when the same two N-terminal glycosylation sites of β2AR are added to the trace amine

13 associated receptor 1 (TAAR1) there is an increase in the surface expression of this receptor which otherwise is normally retained in the ER (Barak et al., 2008). However when the glycosylation sites of the AT1R (Deslauriers et al., 1999) and FSH (Davis et al., 1995) receptors are removed, the receptors are retained in the ER. In contrast, the surface expression of the muscarinic M2 (van Koppen and Nathanson, 1990), histamine H2 (Fukushima et al., 1995) and α1AR (Sawutz et al., 1987) are not affected when the N-glycosylation sites are removed.

Although N-linked glycosylation is the most common glycosylation, receptors can also undergo O-linked glycosylation. However, the precise role and function of O-linked glycosylation for GPCRs is not well understood. While there is no consensus sequence for O- linked glycosylation, it is known to occur within the golgi apparatus where serine and threonine residues are glycosylated (Duvernay et al., 2005). Much as for N-glycosylation, effects of O- glycosylation on receptor function are not universal. For example, the removal of O-linked glycosylation sites in the V2R receptor does not alter the surface expression of that receptor (Sadeghi and Birnbaumer, 1999). In contrast, O-glycosylation is essential for the maturation and surface expression of the δ-opioid receptor (DOR) (Petaja-Repo et al., 2000). Therefore it can be seen that glycosylation is an important post translational modification for most receptors. However much like the ER export and retention motifs, the effects on receptor surface expression are receptor specific and not universal to all GPCRs.

The third most common post translational modification to GPCRs involves palmitoylation. GPCR palmitoylation is a dynamic and reversible process of the addition or removal of palmitic acid to mostly cysteine residues via a thioester bond (Qanbar and Bouvier, 2003). Palmitoylation has been shown to occur early in the maturation process of GPCRs in the ER where palmitoylation is a requirement for proper GPCR targeting to the plasma membrane of several GPCRs (Karnik et al., 1993; Zhu et al., 1995; Schülein et al., 1996; Fukushima et al., 2001; Percherancier et al., 2001). Apart from palmitoylation’s role in surface expression, palmitoylation can act to orientate the C-terminal tail in a position to increase the affinity for arrestin binding and internalization of the receptor. This is clearly seen with the V2R receptor where palmitoylation deficient mutants are internalized much less efficiently upon stimulation of agonists even though their phosphorylation states are similar to the WT receptor (Charest and Bouvier, 2003).

In sum, glycosylation and palmitoylation have large impacts on not only receptor trafficking to the plasma membrane but on the signalling of receptors as well. The effects of these post translational modifications are elegantly reviewed in the following references (Qanbar and Bouvier, 2003; Duvernay et al., 2005)

1.4.3 Molecular Chaperones

As explained in the previous section, some GPCRs require post-translational modifications for proper trafficking to the plasma membrane, such as presence of ER export motifs, or the masking of ER retention motifs. There are a multitude of different GPCR chaperone proteins that can be classed into proteins that bind the C-terminus, intracellular loops, and the N-terminus. For a review of different interactor proteins that promote and increase surface expression see the following reviews (Duvernay et al., 2004; Dong et al., 2007; Dunham and Hall, 2009). For the purposes of this thesis, we will focus on the interactors that are required for the surface expression of GPCRs which we term here as molecular chaperones.

Some receptors require the binding of specific chaperones for proper trafficking to the plasma membrane. The existence of molecular chaperones was postulated based on the observation that certain receptors expressed little to no functional protein when expressed in heterologous systems (McClintock et al., 1997; Couve et al., 1998; Borowsky et al., 2001; Bunzow et al., 2001).

The first class of chaperones are a general class of ER localized chaperones that bind and aid the folding of receptors, these include calnexin, calreticulin and ER heatshock proteins such as Grp78 and Grp94 (Siffroi-Fernandez et al., 2002; Mizrachi and Segaloff, 2004). The function of these chaperones is to promote the correct folding of GPCRs as well as aiding the trafficking of correctly folded receptors to the plasma membrane. For instance calnexin and calreticulin recognize and bind to early glycosylated receptors and promote the N-linked glycosylation of certain receptors (Williams, 2006). On the other hand, heatshock proteins such as Grp78 and Grp94 aid the proper folding of receptors by binding the exposed hydrophobic patches of newly synthesized proteins in the ER (Hamman et al., 1998).

The second class of chaperones are specific for GPCRs and are known as molecular chaperones. The first example of such molecular chaperones is other GPCRs themselves. As

15 described in section 1.4.3 the GBR1 receptor contains an ER retention motif on its C-terminus (Couve et al., 1998). It is upon dimerization with its molecular chaperone, GΒR2, that proper surface expression of the GBR1 receptor is achieved (White et al., 1998), whereby the dimerization of GΒR2 masks the retention signal of GBR1 through a coiled coil interaction of the two C-terminal domains (Margeta-Mitrovic et al., 2000b; Villemure et al., 2005). Similarly the dimerization of α1dAR with α1bAR or α2bAR strongly promotes the surface expression of the α1dAR (Uberti et al., 2003, 2005; Hague et al., 2004). Conversely dimerization can retain receptors in the ER rather than promote surface expression. The clearest examples are the following dominant negative interactions of receptors. For example the heterodimerization of the common DOR Cys-27 with the wild type DOR Phe-27 caused a decrease in surface expression of the WT DOR Phe-27 (Leskelä et al., 2012). This is also seen when a mutated β2AR, that harbours an ER retention motif, is able to significantly decrease the surface expression of a co- expressed WT β2AR (Salahpour et al., 2004).

In addition to GPCRs, there are an increasing number of single transmembrane proteins that act as molecular chaperones for specific receptors. These small proteins act as molecular chaperones but have also been shown to modify receptor signalling as well. The first of these small membrane proteins discovered are the receptor activity modifying proteins 1-3 (RAMPs). RAMPs are single transmembrane proteins that are key regulators of receptor trafficking and signalling. RAMPs act as molecular chaperones for the calcitonin receptor-like receptor (CRLR) where binding with RAMPs enables proper surface expression of the receptor (McLatchie et al., 1998). In addition to the CRLR it has been shown that RAMPs interact and promote the surface expression of several other class B and C GPCRs (reviewed by Hay et al., 2006).

The family of receptor transporting proteins (RTP 1,1s,2-4) are also single transmembrane proteins that act as molecular chaperons for odorant receptors (Saito et al., 2004). REEP1 (receptor expression enhancing protein 1), which is also a single transmembrane protein, also increases the surface expression of odorant receptors but to a lesser extent then RTP1 and RTP2 (Saito et al., 2004). Lastly the melanocortin-2-receptor accessory protein (MRAP) family of single transmembrane proteins bind, traffic, and modify the signalling of all 5 members of the melanocortin receptors (Chan et al., 2009). Based on structure function studies made with RTP1s, it has been proposed that the N-terminus is crucial for the exit of the receptor complex

16 from the ER, their transmembrane domain is important for the trafficking out of the Golgi, and their C-terminus required for interaction with the receptor itself (Wu et al., 2012a).

In summary molecular chaperones are an important aspect of GPCR surface expression where they are required for the surface expression of specific receptors or receptor classes. While a number of specific molecular chaperones have been identified, there remains a list of receptors that do not traffic to the plasma membrane in heterologous systems, potentially requiring yet undiscovered molecular chaperones (ie TAAR1).

1.4.4 Pharmacological Chaperones and Diseases

Pharmacological chaperones (also referred to as pharmacochaperones and pharmacoperones) are compounds that bind and increase the efficiency of receptor folding leading to an increase in total and surface expression. The discovery of pharmacological chaperones came from the study of human diseases that are the result of mutations that result in ER retention and lack of surface expression of the mutant receptor. One of the first diseases to be described is nephrogenic diabetes insipidus (NDI) that results from a variety of mutations on the V2R (Bernier et al., 2004). The mutant receptors are, for the most part, retained in the ER and do not traffic to the plasma membrane. The lack of resulting signalling from these receptors does not allow the kidneys to concentrate urine correctly, resulting in chronic dehydration. It was found that the addition of lipophilic antagonists to heterologous cell lines expressing these V2R mutants rescued their surface expression and allowed for proper V2R based signalling (Morello et al., 2000).

Autosomal dominant retinitis pigmentosa can be caused by one of thirteen point mutations in rhodopsin. These mutations cause the retention of rhodopsin in the ER with no 11- cis-retinal binding (Dryja et al., 1990; Sung et al., 1991; Dejneka and Bennett, 2001). Surface expression of these mutants can be rescued with 11-cis-ring-retinal analog of 11-cis-retinal rescuing the surface expression of these mutant rhodopsins (Noorwez et al., 2003).

Specific mutations in the gonadotropin releasing hormone receptor result in receptor retention in the ER and can result in hypogonadotropic hypogonadism. Interestingly this study was the first to do a screen of compounds to discover novel pharmacological chaperones using this mutant receptor. The result of this screen found a wide range of compounds that act as

17 pharmacological chaperones with differing efficacy in the rescue of surface expression of the receptor (Janovick et al., 2003).

Lastly, heterozygous null mutations in the MC4R receptor cause an imbalance in metabolic homeostasis leading to the early onset of obesity in humans (Martinelli et al., 2011). Treatment with a novel MC4R antagonist causes an increase in the folding and surface expression of the receptor, consistent with pharmacological chaperoning (René et al., 2010).

Although the study of pharmacological chaperones is relatively new, it has been proposed that pharmacological chaperones exist for almost all GPCRs (see Maya-Núñez et al., 2012). Indeed clinical interest in the therapeutic benefits of pharmacological chaperones is on the rise. One interesting example is the use of β2AR antagonists as a long term treatment for asthma (Walker et al., 2011). The rationale behind this treatment is paradoxical where current treatment for asthma involves the acute administration of β2AR agonists that act to relax the muscles of the airway. However the authors propose that adding low doses of the β2AR antagonist nadolol increases the surface expression of the endogenous receptors (presumably through pharmacological chaperones or stabilization of the receptors to the plasma membrane), allowing for long term alleviation of asthma symptoms without the need for β2AR agonists which are known to have adverse effects when used for long term treatment of asthma.

1.5 GPCR Oligomerization

It is well established that many non-GPCR receptors, like receptor tyrosine kinases, exist and function as dimers (Heldin, 1995; Bain et al., 2007). Although early studies with purified rhodopsin showed this receptor existing in ordered oligomers, these were mainly attributed to experimental artifacts and thought to have no physiological function (see reviews Hébert and Bouvier, 1998; Salahpour et al., 2000). However many recent studies have shown the existence of GPCR oligomerization and its importance in vivo and in mediating physiological responses (Angers et al., 2002).

The assembly of dimers can occur as early as in the ER, as part of the maturation of the receptor. Specifically this is the case with the GABA b receptors and the V2R (See section 1.4.3). Indeed these receptors form stable dimers in the ER and are trafficked to the plasma

18 membrane as dimers. However other receptors form oligomers quite transiently while on the plasma membrane via yet unknown mechanisms (please see review Lohse, 2010).

There are several important functional consequences for oligomerization. As described above, the first role of dimerization is the regulation or requirement for the export of GPCRs from the ER (see section 1.5). The second function of oligomerization is the alteration of pharmacological properties of receptors. One such example is the δκ-opioid heterodimer losing the ability to bind to the selective ligands for either of the monomers (Gomes et al., 2000). This is also seen with the co-expression of the μδ-opioid receptors (George et al., 2000). In addition dimerization has also been shown to alter G-protein coupling whereby the dimerization of the D1 and D2 dopamine receptors leads to coupling of the heterodimer to Gαq where individually the D1 and D2 couple to Gαs and Gαi respectively (Lee et al., 2004; Rashid et al., 2007).

Lastly, it has been shown that the GPCR oligomer is the functional receptor unit that binds and signals upon agonist activation. For example the heterodimerization of the GBR1 and GBR2 receptor is necessary for a fully functioning GABAb receptor. By creating a chimeric GBR1/2 receptor, it was shown that the intracellular helical domain of GBR2 is required for G- protein binding and the extracellular domain of GBR1 is required for GABA binding (Galvez et al., 2001). This cooperative binding and signalling seen in the GBR1/GBR2 heteromer has been termed asymmetrical activation where activation of one GPCR affects the other receptor within the dimer complex (Damian et al., 2006; Hugo et al., 2006; Albizu et al., 2010). Further evidence for a functional receptor as an oligomer includes the homodimer of the metabotropic glutamate receptor 2 (mGluR2) being required for the proper binding and signalling of the receptor. While the monomeric mGluR2 is sufficient for G-protein coupling, only dimerization allows the receptor to signal when bound to its endogenous ligand, glutamate (El Moustaine et al., 2012).

Although oligomerization of GPCRs is a relatively new development, it is quite clear that GPCR oligomerization is involved in GPCR regulation and signalling.

1.6 GPCR Endocytic Trafficking

The other important trafficking aspect of GPCRs involves the internalization and desensitization of the receptors. The first step of internalization relies on the uncoupling of the

G-protein upon receptor activation. Once the GPCR is de-coupled, the receptor can undergo phosphorylation in the intracellular loops and C-terminus via GRKs or PKA (Lefkowitz, 1993). The phosphorylation of the receptor promotes the binding of arrestins allowing for the receptor to enter the endocytic pathway for internalization. Once internalized the fate of the GPCR can be one of the following: recycling of the receptor back to the plasma membrane, proteolytic degradation, or mediating alternative signalling pathways. In general receptor desensitization can be characterized into homologous and heterologous desensitization. Heterologous desensitization refers to inactive receptors being phosphorylated and internalized through an agonist independent manner. This phosphorylation is done by the activation of secondary messenger dependent protein kinases (ie PKC) (Lefkowitz, 1993). The term homologous desensitization refers to the requirement of agonist occupation of a receptor for desensitization and will be described below.

1.6.1 G-protein Coupled Receptor Kinases (GRK)

The first stage of GPCR desensitization is through the phosphorylation of specific serine and threonine residues on the third intracellular loop and the C-terminal domain of the GPCR (Premont et al., 1995; Ferguson et al., 1996). This phosphorylation is mediated by the G-protein coupled receptor kinases (GRK) which is the initial step of GPCR desensitization and propagation to the following three pathways. 1) The uncoupling of G-proteins from the receptor through this phosphorylation and subsequent binding of arrestins, 2) endocytosis of the receptor into endosomes and 3) GPCR signaling through G-protein independent mechanisms. The mammalian family of GRKs consists of 7 GRKs where GRK 2, 3, 5, and 6 are ubiquitously expressed while GRK 1 and 7 are expressed selectively in the visual system. Structurally, all GRKs contain three functional domains: N-terminal regulator of G-protein signalling (RGS) homology domain, central catalytic domain, and a C-terminal targeting domain. (for a detailed review of specific GRK domains and their function see (Marchese et al., 2008)). Although phosphorylation is the first step of desensitization, it is not sufficient to completely uncouple the GPCR from the G-protein. This was first reported with the discovery of GRK1 where it was observed that phosphorylated rhodopsin could still bind and signal through transducin (McDowell and Kühn, 1977). Therefore the role of GRKs is necessary to initiate desensitization and internalization of GPCRs but is not sufficient to mediate complete desensitization.

1.6.2 Arrestins

It is the binding of arrestins to the phosphorylated receptor that completes the desensitization of the GPCR by blocking the G-protein binding site as well as mediating receptor endocytosis. This was first reported with the discovery of the visual arrestin (arrestin 1) whereby complete decoupling of rhodopsin from transducin was mediated through the binding of arrestin 1 (Bennett and Sitaramayya, 1988). In general arrestins are intracellular proteins that bind phosphorylated GPCRs and mediate their endocytosis through a clathrin dependent pathway (Ferguson et al., 1996; Goodman et al., 1996). Four arrestin isoforms are expressed in mammals: arrestin 1 -4. It is important to note that arrestin 2 and 3 are also named β-arrestin1 and β- arrestin2 respectively and this will be the notation used from this point forward. Conversely arrestin 1 and 4 will be termed visual arrestins. The key difference between the visual arrestins and β-arrestins lies in the C-terminal tail of the protein. β-arrestins contain two motifs that link the GPCR to the clathrin dependent endocytic machinery. It has been shown that β-arrestins bind with high affinity with clathrin in vitro while visual arrestins do not (Goodman et al., 1996). There exists two classes of receptors based on the β-arrestin binding profile: Class A and B. Class A receptors such as the β2AR preferentially bind β-arrestin2 over β-arrestin1 while Class B receptors such as the V2R bind β-arrestin1 and β-arrestin2 with similar affinities as well as having the ability to interact with visual arrestins (Oakley et al., 2001). Furthermore class A receptors bind β-arrestin more transiently where β-arrestin2 is dissociated from the receptor during endocytosis(see reviews Ferguson, 2001; Luttrell, 2008). Class B receptors on the other hand form a more stable association with the arrestins where the arrestins do not dissociate upon receptor endocytosis (Zhang et al., 1999a). In the case of class A receptors, transient interaction with β-arrestin2 allows for the dephosphorylation and recycling of the receptors to the plasma membrane. Conversely, class B receptors recycle less efficiently since they have stable interactions with arrestins (see section 1.6.2). The structural determinant for the stability of arrestin interaction is found within the serine and threonine clusters of the C-terminal tail. Indeed switching the C-terminus of the β2AR with that of the V2R diminishes the recycling of β2AR. Conversely the V2R receptor recycling is increased with the C-terminal tail of β2AR, a typical class A response (Oakley et al., 1999, 2001). The stable or transient β-arrestin association with GPCRs has been shown to be mediated through different phosphorylation patterns by different GRK isoforms. For instance while the β2AR is traditionally a class A receptor, over expression

21 of GRK5 and 6 leads to a stable β2AR -βarrestin complex during internalization, a typical class B response (Shenoy et al., 2006).

In addition to arrestin’s role in mediating endocytosis of GPCRs, it is now well established that internalized GPCRs can still signal through arrestin mediated pathways in a G- protein independent manner. The GPCR – arrestin signalling pathway has now been shown to bind a wide variety of proteins including, kinases, small GTPases, guanine nucleotide exchange factors, E3 ubiquitin ligases, phosphodiesterases, and transcription factors (Reiter and Lefkowitz, 2006; Gesty-Palmer and Luttrell, 2008; Luttrell, 2008; Rajagopal et al., 2010). Indeed the evidence for the biological significance of arrestin signalling has been increasing. For instance it has been postulated that the anti-psychotic effects of lithium are primarily due to its ability to inhibit glycogen synthase kinase 3, a downstream effector protein of the dopamine D2-β-arrestin signalling pathway (Beaulieu et al., 2008). Furthermore what is arguably more interesting is the observation that ligands for GPCRs can bias the signalling of GPCRs through either the G- protein dependent pathway or the β-arrestin pathway. This type of signalling is termed, functional selectivity or biased signaling (Gesty-Palmer and Luttrell, 2008; Luttrell and Kenakin, 2011).

Although arrestin mediated endocytosis in clathrin coated pits is the most common form of receptor endocytosis, some GPCRs can also internalize through an arrestin independent manner. These receptors internalize using a C-terminal motif that interacts with adaptor protein 2 (AP2) found in clathrin coated pits, resulting in clathrin dependent endocytosis (Blanpied et al., 2002; Santini et al., 2002; Scott et al., 2002). The motifs includes two tyrosine based motifs YXXϕ and YXXXϕ (where ϕ represents a bulky hydrophobic amino acid), and the more common dileucine motif. For example the protease activated receptor 1 (PAR1) is a receptor that internalizes through this mechanism. Mutation of the YXXϕ domain to a aXXa domain in this receptor completely impairs its internalization upon agonist stimulation (Paing et al., 2004). The YXXXϕ motif is found in the thromboxane A2 β (TPβ) receptor and is responsible for the tonic internalization of the receptor (see section below) (Parent et al., 2001). The dileucine motifs are present in multiple GPCRs such as the β2AR (Moore et al., 2007). Although the β2AR undergoes arrestin mediated endocytosis, the mutation of the dileucine motif decreases the amount of receptors internalized upon agonist stimulation indicating that two mechanisms of internalization are occurring (Gabilondo et al., 1997). While these mutations reduced ligand

22 induced internalization, the signalling through the receptor was otherwise not affected (Gabilondo et al., 1997; Kohout et al., 2001). Although the dileucine motif is important for internalization of certain receptors, it might also play a role as an ER export signal (see section 1.4.1). It is clear that internalization is a complex process where dynamics between arrestin independent and dependent internalization are currently unknown (Magalhaes et al., 2012).

1.6.3 Tonic/Constitutive Internalization

Receptors can also undergo tonic internalization in the absence of any ligand stimulation following a distinct mechanism. For example the TPβ internalizes through the arrestin pathway when stimulated with an agonist while tonic internalization is mediated through the YXXXϕ domain mentioned above (Parent et al., 2001). Beyond the regulation of receptor homeostasis, it is not yet known what role tonic endocytosis plays in the signalling pathways mediated by GPCRs.

1.6.4 Endocytic pathway

Once the receptors are internalized, they are either recycled back to the plasma membrane or targeted for degradation. The stability of β-arrestin interaction with the receptor acts as the major contributor as to whether or not the receptor is recycled back to the plasma membrane. For example the class A receptor β2AR, has a rapid dissociation with β-arrestin upon internalization through clathrin coated pits, allowing for the receptor to enter a more acidic endosomal compartment that leads to dephosphorylation of the receptor and its recycling back to the plasma membrane (Pitcher et al., 1995). In contrast, class B receptors like the V2R internalize in complex with β-arrestins slowing the dephosphorylation of the receptor which leads to subsequent proteolytic degradation of the receptor (Oakley et al., 1999). Therefore it appears that the stability of receptor-β-arrestin interaction determines whether the receptor is recycled to the plasma membrane or degraded (Luttrell and Lefkowitz, 2002). Further regulation of GPCRs in the endosome has been found with the NPXXY motif on the cytoplasmic end of the 7th transmembrane domain which contributes to the endocytosis of many GPCRs (Gripentrog et al., 2000; Bouley et al., 2003; Kalatskaya et al., 2004). Although less understood, cytoplasmic C- terminal motifs such as the PDZ (PSD-95 protein), DLG (Drosophila discs large protein) and ZO-1 (zonula occludens-1 protein) motifs regulate endocytic sorting and are present in a large number of GPCRs (see review by Marchese et al., 2008).

In addition to the motifs present on the receptors, post translational modification of arrestins can also be important for internalization of receptors. For instance ubiquitination of β- arrestin 2 by the E3 ubiquitin ligase MDM2 is essential for the internalization of β2AR (Shenoy et al., 2001).

Lastly if the receptor is not recycled to the plasma membrane, the receptor-arrestin complex is ubiquitinated and subsequently targeted to the lysosome for degradation. In most cases, targeting of the receptor-arresting complex to the lysosome occurs most commonly through ubiquitination however ubiquitination independent targeting has also been reported (see review Marchese and Trejo, 2013).

In summary, the endocytic pathway is important in the long term regulation of GPCR signalling. Once internalized, the receptor is sorted through the recycling or degradation pathways. Recycling of the receptor is thought to be the main mechanism for receptor resensitization while receptor degradation is the main mechanism for down regulation of the receptors (Hanyaloglu and von Zastrow, 2008). Furthermore internalized receptors can initiate other signalling pathways in a G-protein independent manner.

1.7 Summary of Assays for measuring surface expression of GPCRs

The concept for quantification of surface receptors is a relatively simple idea, whereby only receptors on the plasma membrane of the cell are detected. The most widely adopted assays for quantification of surface expression use either hydrophilic ligands or antibodies in order to quantify cell surface expression of GPCRs. However, as will be discussed later in this section, there are several newly developed assays for quantification of cell surface receptors where the primary purpose of these assays has been miniaturization and their adaptation and use for high throughput approaches.

The first assays used to quantify surface expression of GPCRs utilized hydrophilic radioligands that do not cross the plasma membrane (Lohse et al., 1990; Green and Liggett, 1994; Mialet-Perez et al., 2004). In the absence of radiolabelled hydrophilic ligands, cell fractionation was used to isolate the plasma membrane from whole cell lysates (Lohse et al., 1990). Once isolated, surface receptors were quantified using radioligands and autoradiography (Jockers et al., 1996, 1999) or immunoblotting (Jockers et al., 1999; Lee et al., 2000). Given the

24 limited availability of radioactive hydrophilic ligands as well as the time consuming process of cell fractionation, there has been a transition to simpler and more efficient assays over the last 20 years.

Currently, the gold standard for the quantification of receptors at the plasma membrane is through antibody mediated assays (flow cytometry and ELISA) or biotinylation. These methods are well established and have been used in multiple publications looking at surface expression of GPCRs. The following are examples of studies using flow cytometry (Ramprasad et al., 1996; Jockers et al., 1999; Morello et al., 2000; Compton et al., 2002), ELISA (Salahpour et al., 2004; Rochdi et al., 2010; Lan et al., 2011), and biotinylation (Ramprasad et al., 1996; Ray et al., 1998; Petaja-Repo et al., 2000; Wüller et al., 2004). ELISA and flow cytometry are accomplished using live whole cell preparations expressing recombinant GPCRs with the addition of N-terminal epitopes. Exogenous epitopes are used due to the availability of selective high affinity primary antibodies towards them. The basic steps for both flow cytometry and ELISA include the following: Blocking, probing with 1˚ antibody, fixing the cells, and adding 2˚ antibody. In the case of an ELISA the 2˚ antibody is a horseradish peroxidase (HRP) conjugated antibody where HRP enzymatically converts a colourometric compound (o-Phenylenediamine dihydrochloride) that can be quantified using a spectrophotometer (for procedure reference please see Salahpour et al., 2004). For flow cytometry the secondary antibody is a fluorophore conjugated antibody that can be detected and quantified by a flow cytometer (Jockers et al., 1999). Both of these assays can be done within a day and have been shown to produce reliable and reproducible results. In addition, once optimized, these assays can be miniaturized to at least 96-well plate format, therefore increasing their throughput. Although there have been no attempts to optimize these assays for high throughput screening of surface expression, ELISA and flow cytometry have been optimized for other systems to the point where they are suitable for high throughput screening. Examples include high throughput discovery of protein biomarkers for ELISA and identification of small molecule inhibitors for proteins using flow cytometry. For reviews of this topic please see (Jin and Zangar, 2010) and (Sklar et al., 2007) for ELISA and flow cytometry respectively. The main advantages of flow cytometry and ELISA stem from the increase in robustness and throughput from previous generation surface expression assays (see above) where these assays can be completed in a 96 well plate compared to cell fractionation and radioligand binding. Furthermore flow cytometry can be used to measure

25 multiple labeled receptors versus just one for the ELISA. The disadvantage of flow cytometry and ELISA are the relative low throughput of these assays when compared to new surface expression assays as described below (section 1.7.1-1.7.3).

The third standard for measuring surface expression is the use of cell surface biotinylation. Biotinylation allows for the purification of surface proteins by taking advantage of the high affinity binding of biotin to streptavidin. The method requires the biotinylation of all surface proteins of a live cell. Once biotinylated, the cells are lysed and run through a streptavidin column, binding all biotin labelled cell surface proteins. After several washes, the column is eluted with the addition of free biotin. The eluted fractions are then run on an SDS- PAGE gel where immuno blotting is subsequently used to quantify the protein of interest. Unlike an ELISA or flow cytometry, biotinylation can be used for native proteins if a suitable antibody is available. However, biotinylation cannot currently be done in a high throughput manner because of the affinity column and the long wash and incubation steps required.

Within the past 5 years several other assays have been introduced that significantly increase the efficiency, cost, and robustness of surface expression measurements. The first method is the conjugation of the primary antibody with a quantum dot (Fichter et al., 2010). Quantum dots are inorganic semiconductors that can undo go fluorescence, like organic fluorophores, but have distinct advantages over traditional organic fluorophores. They can be engineered, based on the size of the quantum dot, to have specific excitation and emission spectra as well as a wide band gap increasing the signal to noise ratio. In addition, quantum dots are immune to photobleaching and are 10-20 times brighter than organic fluorophores (For a review of quantum dots for cellular imaging please see Alivisatos et al., 2005). By using quantum dots, the signal to noise of the assay when using flow cytometry is greatly improved.

1.7.1 Fluorogen Activating Protein Biosensor

The second assay that improves upon traditional flow cytometry uses a fluorogen activating protein (FAP) based biosensor. FAP is a relatively small 200 amino acid protein that upon binding of a fluorogen increases the fluorescence of the fluorogen dramatically as part of a non-covalent reaction (Szent-Gyorgyi et al., 2008). Although this FAP biosensor can be conjugated with antibodies for cell surface labeling (Szent-Gyorgyi et al., 2008), the FAP biosensor can actually be fused on the N-terminus of GPCRs (Fisher et al., 2010). This method

26 requires the use of cell impermeable fluorogens that selectively label cell surface receptors. This assay improves upon traditional flow cytometry by removing the need for antibodies thereby improving the signal to noise and throughput of the assay. Recently, the FAP assay was optimized for high throughput screening of ligands that promote internalization of the β2AR (Wu et al., 2012b).

1.7.2 Internalization Assays

In addition to the approaches developed for measuring surface expression, assays have also been designed to specifically monitor receptor internalization rather than total surface expression. These assays can be split into two separate types. The first is the binding of β- arrestin to the GPCR, while the second assay is the recruitment of receptors to endosomes. For binding of β-arrestins to GPCRs, two distinct techniques have been developed. The first is the use of bioluminescence resonance energy transfer (BRET) whereby the β-arrestin is tagged with either the donor or acceptor BRET partner (or both acceptor and donor Charest et al., 2005). The assay relies on measuring the energy transfer between β-arrestin and the GPCR upon agonist activation (for a review see Salahpour et al., 2012). The second assay utilizing β-arrestin is a complementation assay whereby fragments of β-galactosidase are found on the C-terminus of the GPCR and on the β-arrestin (Hammer et al., 2007). The binding of β-arrestin completes the β- galactosidase enzyme whereby a β-galactosidase substrate can then be added allowing for a quantifiable signal. The advantages of these β-arrestin assays are their simplicity and ability to be used in high throughput screening (Hamdan et al., 2005; Hammer et al., 2007).

The second technique looking at receptor localization to endosomes relies on pH sensitive fluorescent proteins (phlourins) that will have altered fluorescent properties due to the lower pH of endosomes (Geisow and Evans, 1984). The first assay using phlourins has traditionally been used for imaging GPCRs in endosomes rather than quantification of receptor internalization (Miesenböck et al., 1998; Mahon, 2011; Yudowski and von Zastrow, 2011). However this technique has evolved to allow for quantification of internalization using a N- terminal coil-coiled tag probes (Yano et al., 2012). These probes are different than typical phlourins due to the decrease in protein size on the N-terminus of the GPCR as well as allowing for a wide range of conjugated pH sensitive fluorophores to be used (Takeda et al., 2012). Lastly by tagging a β-Galactosidase fragment with the FYVE domain, of the endosomal protein

27 endofin, receptor localization within endosomes can be quantified in a similar fashion to the β- arrestin complementation assay (Hammer et al., 2007). The advantage of these assays compared to the β-arrestin assays, is the ability to quantify the amount of receptors internalized to endosomes.

1.7.3 N-terminal GPCR Fusion Tags

Next, a multitude of fusion proteins have been developed to observe specific compartments in cellular imaging. These fusion proteins, or tags, rely on small fluorescent organic molecules covalently linked to the tags through enzymatic ligation (Sun et al., 2011). Having compounds that are sensitive to their chemical environment (eg. cell permeable compounds) can allow for the specific labelling of fusion proteins in different cellular compartments. Although various examples for each tag exist, only the SNAP and CLIP tags have been developed and optimized to measure surface expression (Doumazane et al., 2011). The SNAP tag is derived from the 20kDa DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) that covalently binds O6-benzylguanine derivatives that are fluorescently labeled (Juillerat et al., 2003). The CLIP tag is a derivative of AGT that selectively binds O6- propylguanine analogs (Gautier et al., 2008). Using different combinations of CLIP and SNAP tags in addition to cell impermeable substrates, surface expression can be quantified using this technique (Maurel et al., 2008; Zwier et al., 2010; Doumazane et al., 2011; Ward et al., 2011). In addition the substrates for both the SNAP and CLIP tags can be modified to contain two fluorophores allowing for FRET (Maurel et al., 2008). By utilizing FRET, the signal to noise of this assay is dramatically increased to such a point that SNAP-tagged receptors can be used for high throughput screening (Haruki et al., 2012). Although assays using SNAP-tagged fusion proteins have yielded a Z’ > 0.7 (see section 4.6 for definition of Z’), quantification of surface expression of SNAP tagged GPCRs in a high throughput manner has not yet been optimized.

In this thesis, we describe a new assay to quantify surface expression of GPCRs. We utilize β-lactamase (βlac) fused to the N-terminus of GPCRs, as our reporter for this assay (Figure 1.2). In addition we utilize the chromogenic β-lactamase substrate nitrocefin in our assay for quantification (Figure 1.3). Nitrocefin is a well validated substrate for β-lactamases and is the main substrate used when screening for β-lactamase activity. Nitrocefin on its own is a coloured substrate that has a max absorption at 390 nm (yellow). Upon the cleavage of the β-lactam ring

28 by β-lactamase, there is a shift in the peak absorption to 486 nm (red) (O’Callaghan et al., 1972) (Figure 1.2).

1.8 β-lactamase Assay

Although the use of β-lactamases as reporters for proteins is not a new concept (Moore et al., 1997; Watanabe et al., 2011), we describe a new assay that utilizes βlac-GPCR fusion receptors to quantify the surface expression levels of GPCRs (Figure 1.2). While others have used β- lactamase to differentiate the localization of specific proteins in the cell (Watanabe et al., 2011), we are the first to utilize N-terminal βlac on GPCRs to quantify the surface expression on the plasma membrane in a robust manner. Since βlac is a relatively large protein (~30 kDa), it is possible that the function of the βlac tagged receptor could potentially be affected. However it has been shown in the literature that other bulky N-terminal tags (ie SNAP-tag) do not affect the expression or function of multiple GPCRs (Maurel et al., 2008) and our own results indicate that for the GPCRs tested, βlac fusion does not affect receptor activity.

β-lactamases are a family of enzymes that are expressed in prokaryotes and confer a form of antibiotic resistance (review of the last 30 years of β-lactamase inhibitors see Drawz and Bonomo, 2010). β-lactam based antibiotics act on the penicillin binding proteins (PBP) that inhibit the transpeptidase action of PBPs which are necessary for the cell wall synthesis of prokaryotes (Zapun et al., 2008). Therefore β-lactamases act as enzyme mimics of PBPs and are serine hydrolases that acylate the β-lactam ring of antibiotics (Minasov et al., 2002). The activated serine of the β-lactamase enzyme acylates the β-lactam containing compound, whereby the active site of the enzyme is regenerated following the deacylation of the enzyme with activated water (figure 1.4). We chose to use β-lactamase as our reporter due to a lack of an eukaryotic homolog resulting in no background hydrolysis of nitrocefin (Moore et al., 1997). We also chose to use nitrocefin as the substrate for our assay due to its commercial availability and its thorough characterization in the literature as a suitable substrate for β-lactamase (Jones et al., 1982; Zygmunt et al., 1992; Moore et al., 1997; Bouillenne et al., 2000; Tan et al., 2003). Apart from nitrocefin, there are other compounds that can act as quantifiable substrates for β- lactamase. For example alternative chromogenic β-lactamase substrates have been described in the literature. The compounds PADAC (Jones et al., 1982) and CENTA (Bebrone et al., 2001) have been shown to have similar kinetics to nitrocefin and would also be suitable ligands for our

Figure 1.2: Schematic of the βlac assay. The βlac assay uses an N-terminal βlac-GPCR fusion protein that is transfected into cells. If there is no surface expression of this construct (left panel) then the addition of the cell impermeable nitrocefin will cause no colour change. If the construct is expressed on the plasma membrane, the extracellular βlac can now cleave nitrocefin causing a shift in absorbance from 390nm (yellow) to 486nm (red).

Figure 1.3: Nitrocefin as a chromogenic substrate for the βlac assay. Nitrocefin is a β-lactam compound that absorbs light at λ=390nm natively. Upon hydrolysis of the β-lactam ring by β- lactamase the absorbance shifts at λ=486 nm.

31 assay. Unfortunately none of these two alternatives is commercially available. In addition it has been shown that ampicillin and its esterified analog bacampicillin; can be chemically conjugated with cationic fluorophores to become suitable fluorescent β-lactamase substrates (Watanabe et al., 2011). By using ampicillin and its pro-drug variant, cell permeant and cell impermeant fluorophore conjugated β-lactam analogs were created. However these β-lactam analogs are not well characterized in the literature and are only available through chemical synthesis. Lastly there exists a commercially available FRET based cephalosporin analog substrate for βlac (Zlokarnik et al., 1998; Rukavishnikov et al., 2011). However this substrate is cell permeable and therefore not suited for quantifying surface expression of GPCR. The kinetics of nitrocefin on our Class A β-lactamase (TEM-1) has been reported with a Km and Kcat of 52μM and 930s-1 respectively (Bouillenne et al., 2000). In addition, nitrocefin is impermeable to polymer based liposomes (Städler et al., 2009), however plasma membrane permeability has not yet been experimentally evaluated.

In summary we have developed a novel assay for GPCR surface expression quantification. This assay is sufficiently different from current assays on the market by using a chromogenic substrate that is commercially available. In this thesis, we aimed at validating this novel assay against ELISAs looking at various conditions that affect surface expression of prototypical GPCRs.

Figure 1.4: Mechanism of action for a class A β-lactamase. (1) The β-lactam carbonyl group undergoes nucleophilic attack by the activated ser70, resulting in an acylation intermediate (2). (3) Protonation of the β-lactam nitrogen leads to cleavage of the C-N bond and formation of the covalent acyl-enzyme (4). (5) Attack by a catalytic water leads to a high-energy deacylation intermediate, with subsequent hydrolysis of the bond between the β-lactam carbonyl and the oxygen of Ser70. Deacylation regenerates the active enzyme and releases the inactive β-lactam (Drawz and Bonomo, 2010)

Chapter 2 Materials and Methods

2.1 Reagents

Nitrocefin (BD Biosciences) was dissolved in DMSO at a concentration of 10mM. Isoproterenol (Sigma) was dissolved in PBS containing 170μM ascorbic acid. Alprenolol and propranolol (Sigma) were dissolved in PBS. Mouse anti-HA primary antibody (12CA5 hybridoma), anti- mouse HRP conjugated (Cell Signaling Technology), and alexfluor-680 anti-mouse conjugated (Invitrogen) secondary antibodies were diluted in PBS containing 1% BSA.

2.2 Plasmid Construction

The cDNA expression vectors for human GBR1 and β2AR were provided by Dr. Michel Bouvier (Salahpour et al., 2004; Villemure et al., 2005). The cDNA expression vector for human GBR2 was obtained from Missouri S&T cDNA.

The β-lactamase sequence was cloned from the ampicillin resistance gene within the pcDNA3.1 plasmid, with the restriction sites HindIII and AscI at 5’ and 3’ of the βlac respectively. The βlac was further modified by the additions of a chicken α7 nicotinic receptor signal sequence (SS) [9], and an HA epitope at the N-terminus, yielding the following cDNA: SS-HA-βlac. The βlac construct was subsequently cloned into the multiple cloning site of the pcDNA3.1 plasmid. A 5’ Asc I and 3’ Not I restriction site were added to the GBR1 and β2AR. These cDNA constructs were then ligated into the plasmid in frame with the βlac creating SS-HA-βlac-β2AR and SS- HA-βlac-GBR1.

2.3 Cell Culture

HEK 293 and HEK 293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Wisent) and supplemented with 10% FBS (Wisent), 100 U/ml penicillin and 100µg/ml streptomycin. HEK 293 cells stably expressing SS-HA-βlac-β2AR or SS-HA-βlac-GBR1 were further supplemented with 1µg/ml puromycin. All cells were kept at 37˚C and 5% atmospheric

CO2.

2.4 Generation of Stable Cell Lines and Transient Transfections

Cells (2x106 cells) were seeded into 10cm tissue culture plates. The following day, cells were transfected with 3 µL of polyethylenimine (1mg/mL) (Polyscience Inc) per µg of plasmid DNA. For transient transfections, cells (HEK 293T) were seeded 24h post-transfection for experiments. For stable cell line creation (HEK 293), media was replaced with the proper selection antibiotic 24 hours post transfection. Clonal cell lines were generated by picking individual colonies and expression was confirmed by western blot. For ELISA and βlac experiments, 1x105 cells were plated into individual wells of a poly-D-lysine coated 48 well plate.

2.5 Western Blotting

Clonal cell lines for the SS-HA-βlac-β2AR and SS-HA-βlac-GBR1 were lysed with the addition of RIPA buffer (25mM Tris-Hcl, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1%SDS. 1.5ug/ml aprotinin, 10ug/ml pepstatin A, 10ug/ml leupeptin, 0.25mM PMSF) on ice. The supernatant was collected from the lysates through centrifugation at 15 000 RPM at 4°C. Protein concentration was quantified using the BCA protein assay (Pierce). Samples were heated to 95°C in the presence of β-mercaptoethanol. 30ug of protein was resolved on a 7.5% SDS- polyacrylamide gel and subsequently transferred to a polyvinyl fluoride membrane. Protein expression levels were determined by immunoblotting using the monoclonal anti-HA antibody. Immunoreactivity was detected using the LiCor Odyssey infrared imaging system.

2.6 βlac-β2AR Immunofluorescence

SS-HA-βlac-β2AR stable cell lines were plated at 1x106 cells per well in a 6-well plate containing glass microscope cover slips. 24 hours after the cells were plated, the wells were washed with PBS and blocked with PBS containing 1% BSA and kept on ice. Primary antibody (1:1000) was then incubated for one hour on ice. The primary antibody was removed and warm cell media was added. Cells were then treated for 30 minutes with vehicle or 10μM isoproterenol. The media was then aspirated and the plate washed with PBS. The cells were then fixed with 4% PFA for 15 minutes and subsequently blocked at room temperature with PBS with 1% BSA for 30 minutes. A fluorescent secondary antibody (1:5000) was subsequently added and incubated for 30 minutes. The cells were then washed 3 times with PBS and 1% BSA with a

35 final wash of PBS. The cover slips were then removed and mounted onto microscope slides using Vectashield mounting media (Vector Laboratories). Images were acquired using the eclipse 80i fluorescent microscope (Nikon).

2.7 βlac-β2AR Functional Assay using BRET EPAC cAMP Biosensor

HEK293 cells stably expressing the BRET EPAC cAMP biosensor (Barak et al., 2008) were transfected with 50ng of SS-HA-βlac-β2AR or HA- β2AR and seeded into 96-well plates (1x105 cells) 24 hours post transfection. After 24 hours incubation, cells were washed once with PBS. Coelenterazine H (5 μM, final conc) was added and the plate was incubated for 5 minutes in the dark, after which isoproterenol (1x10-14 – 1x10-4M) was added. Each well was read once every 5 minutes in the Mithras luminometer (Berthold Technologies).

2.8 βlac Assay

Nitrocefin was first diluted to a final concentration of 100 μM in PBS. After drug treatment the cells were washed once with PBS and, after removal of the PBS wash, 200 μL of the nitrocefin solution was added to each well. Immediately after the addition of the nitrocefin solution, absorbance for each well was read kinetically once every minute for 30 minutes at 486 nm using the EPOCH microplate spectrophotometer (Biotek). The rate of reaction (slope of the curve in the linear range) was taken as the readout for this assay

2.9 ELISA

All ELISAs were performed as described previously (Lavoie et al., 2002). Briefly, 24 hours after plating the cells and after drug treatment, plates were washed with PBS and blocked with PBS containing 1%BSA and kept on ice. Primary antibody (1:1000) was then incubated for one hour on ice. The cells were then fixed with 4% PFA for 15 minutes and subsequently blocked at room temperature with PBS with 1% BSA for 30 minutes. Secondary antibody (1:1000) was added and incubated for 30 minutes. The cells were washed 3 times with PBS and 1% BSA with a final wash of PBS where the HRP substrate Sigmafast OPD (Sigma) was subsequently added. After 30-45 minutes of substrate incubation, the reaction was stopped with the addition of 3M HCl. The supernatant was then transferred to a 96 well plate and absorbance was read using the EPOCH microplate spectrophotometer (Biotek) at 492 nm.

2.10 βlac-β2AR Agonist Studies

SS-HA-βlac-β2AR cells were seeded at 1x104 cells (βlac assay) or 1x105 cells (ELISA) per well in poly-D-lysine coated 48 well plates 24 hours prior to experimentation. Isoproterenol was weighed out the day of the experimented and dissolved in PBS containing 170μM ascorbic acid where the doses of isoproterenol were diluted to 100x solutions. To determine the time course of internalization a dose of 10μM of isoproterenol every 5 minutes in triplicate. The dose response of internalization was done by the addition of individual doses (triplicates) of isoproterenol (1x10-11 - 1x10-4 M) followed by incubation at 37°C for 30 minutes. Both the βlac and ELISA were done as stated in section 2.8 and 2.9 for the time course and dose response for SS-HA-βlac- β2AR internalization.

2.11 βlac-β2AR Antagonist Studies

SS-HA-βlac-β2AR cells were seeded at 1x104 cells per well in poly-D-lysine coated 48 well plates 24 hours prior to experimentation. Internalization blocking assays were done by treatment with 30 minute treatment with individual doses (triplicates) of alprenolol or propranolol (1x10-10 - 1x10-4 M) followed by 30 minute incubation with 10μM isoproterenol. Pharmacological chaperoning experiments were done with overnight incubation of individual doses of either alprenolol or propranolol (1x10-10 - 1x10-4 M). The βlac assay was done as stated in section 2.8 for both the blocking and pharmacological chaperoning experiment.

2.12 βlac-β2AR Z’ Determination for Agonist Induced Internalization

SS-HA-βlac-β2AR cells were seeded at 5x103 per well in a 96 well plate 24 hours prior to experimentation. Isoproterenol was weighed out the day of the experiment and dissolved in PBS containing 170μM ascorbic at a 10X concentration of 100μM. Half the plate was stimulated by isoproterenol (10μM final) as the positive control and vehicle was added to the other half of the plate as the negative control. The βlac assay was done as stated in section 2.8 for Z’ determination.

2.13 GBR1 Molecular Chaperoning Studies

SS-HA-βlac-GBR1 stable cell lines were transfected with empty vector or an increasing amount of GBR2 and subsequently plated into a 48 well plate at a density of 1x105 cells per well 24

37 hours post transfection. A βlac assay or ELISA was done 48 hours post transfection as stated in sections 2.8 and 2.9.

2.14 Data Analyses

Data analyses were performed with Graphpad Prism 5.01 (Graphpadsoftware inc). Linear regression was performed on βlac data to determine the slope of the curve for comparisons.

The Z’ factor is a measure of the quality of an assay that takes into account both the signal dynamic range and variation of data. The Z’ was determined using the following equation(Zhang, 1999):

(1) Z’=1-(3σc+ + 3σc-) / (|μc+ - μc-|)

Where μc represents the mean and σc the standard deviations of the controls, where the (+) and (-) denote positive (isoproterenol) and negative (vehicle treated) controls respectively.

Dose response curves were fitted with the following equation:

(2) Y = Ymin + (Ymax – Ymin) / (1 + 10[(pEC50 – logX ) * Hill Slope])

Y represents %response with Ymin and Ymax being the minimum and maximum response respectively. pEC50 is the negative log of the molar concentration to yield 50% maximal response and logX is the molar concentration for response Y. Hill slope represents the degree of the slope within the linear portion of the sigmoidal graph.

One-way ANOVA with Bonferroni correction post-hoc was used to determine the differences between data sets.

Chapter 3 Results

In order to demonstrate the utility of the βlac assay two well characterized GPCRs were used: β2AR and GBR1. It was first determined that the trafficking and signalling of the N- terminal βlac tagged β2AR and GBR1 was not impaired. Using these two receptors the βlac assay was quantitatively compared to the well established ELISA approach for agonist induced internalization of the β2AR and the surface expression of the GBR1. In addition the βlac was also used to determine the Z’ of β2AR internalization, antagonist blocking of agonist induced internalization, and pharmacological chaperoning.

3.1 Generation of the βlac Plasmids and Stable Cell Lines Expressing βlac- GPCR Fusion Constructs

The βlac vector was originally created by Ali Salahpour/Stephane Angers where the Ampr gene was cloned from the pcDNA vector and added in frame to the 3`end of the ORF. In addition, to facilitate the proper plasma membrane targeting of receptor constructs, the chicken α7 nicotinic receptor signal sequence (SS) was added 5`to the βlac sequence. Furthermore, an HA epitope was also added in frame between the signal sequence and the βlac. The GBR1 and β2AR cDNA were cloned with the appropriate restriction sites and added to 3`to the βlac. DNA Sequencing confirmed that the receptor sequences were in frame to the βlac, therefore generating the two following constructs (SS-HA-βlac-β2AR and SS-HA-βlac-GBR1).

Stable HEK293 cell lines were generated by transfecting the cells with either the SS-HA- βlac-β2AR or SS-HA- βlac-GBR1. After selection, monoclonal cell lines were generated from evaluating individual colonies. Western blotting was used to confirm the expression of the βlac- receptor construct (Figure 3.1). Clone 13 of the GBR1 was chosen due to its high expression level while clone 1 was the only clone that was isolated for the β2AR.

3.2 Trafficking and Signalling of the βlac-β2AR

It is always a concern that the addition of a 30kDa tag (in this case βlac) to the N-terminus of a GPCR can affect the function of the receptor. However, it was previously shown

Figure 3.1: Western blot of stable clonal cell line expression levels of SS-HA-βlac-GBR1 and SS-HA-βlac-β2AR. Stable cell lines for the constructs SS-HA-βlac-GBR1 and SS-HA-βlac- β2AR were generated with individual colonies expanded. Mock (non-transfected cells), SS-HA- βlac-GBR1 clones 12-16, and SS-HA-βlac-β2AR clone 1 were lysed in RIPA buffer and boiled at 95°C in the presence of β-mercaptoethanol. 30μg of protein was loaded for each clone in an SDS-PAGE gel and transferred overnight. Blanks represent wells that have not been loaded with protein. Mouse monoclonal anti-HA primary antibody was added with visualization of the blot by alexafluor-800 secondary antibody. The expected bands for the SS-HA-βlac-β2AR and SS- HA-βlac-GBR1 were ~70kDa and ~140kDa respectively. The blot was visualized using the LiCor Odyssey infrared imaging system. SS-HA-βlac-GBR1 clone 13 was chosen due to being the highest expressing clone. Clone 1 of the SS-HA-βlac-β2AR was confirmed to express the construct. The bands did not migrate at these molecular weights due to the boiling of the samples causing the aggregation of the receptors. The low molecular weight bands could be degradation products of the receptors.

40 that the addition of the SNAP-tag (also 30kDa) to the N-terminus of other GPCRs did not affect receptor function (Maurel et al., 2008). The trafficking of the SS-HA-βlac-β2AR receptor for both surface expression and internalization was assessed using immunofluorescence. In addition, the functionality of SS-HA-βlac-β2AR receptor was investigated in a functional assay utilizing a BRET cAMP biosensor EPAC.

Trafficking of the SS-HA-βlac-β2AR was assessed with the use of immunofluorescence (Figure 3.2). Cells were first labelled with anti-HA primary antibody on ice, and subsequently either treated with vehicle or 10μM isoproterenol. In the absence of agonist (Figure 3.2A) the SS-HA-βlac-β2AR had a uniform membrane distribution consistent with surface expression at the plasma membrane (Kim et al., 2008). Stimulation with a dose of 10μM isoproterenol (Figure 3.2B) resulted in receptor internalization marked by the redistribution of receptors to intracellular endosomes consistent with agonist induced internalization (Cao et al., 2005). Therefore the addition of the βlac to the N-terminus of the β2AR does not affect the surface expression nor internalization of the receptors and the results from our experiments (Figure 3.2) are similar to what has been reported in the literature (Cao et al., 2005; Kim et al., 2008).

Secondly, to verify that the function of β2AR was not hindered by the addition of the βlac to the N-terminus, we evaluated the ability of β2AR to stimulate cAMP production. For this we used a previously described EPAC-BRET cAMP biosensor to quantify B2AR function (see section 4.3 for description of this biosensor). Briefly, HEK293 cells stably expressing Rluc-EPAC-YFP were transfected with 50ng of either SS-HA-βlac-β2AR or HA-β2AR and stimulated with isoproterenol. We hypothesized that the two transfected receptors would show distinct pharmacological properties compared to the β2AR that are endogenously expressed in HEK293 cells (see section 4.3). First, the dose response of isoproterenol response in the transfected cells would be left shifted compared to mock cells that endogenously express β2AR (Zhong et al., 1996). Therefore by comparing the dose response of both the SS-HA-βlac-β2AR and HA-β2AR, the effects of the βlac on the signalling of β2AR can be quantified. As seen in figure 3.3A and B, transfection of 50ng of both SS-HA-βlac-β2AR and HA-β2AR shifted the isoproterenol dose response to the left compared to the β2AR signalling observed in mock cells (empty vector transfected cells). Indeed the EC50 of isoproterenol in both the SS-HA-βlac-β2AR and HA-

β2AR, 0.69±0.5 and 0.36 ±0.3 nM respectively, is 170 times lower than EC50 measured in the

Figure 3.2: Immunofluorescence of HEK293 cells stably expressing SS-HA-βlac-β2AR. Cells were seeded in a 6 well plate containing a glass coverslip. Mouse monoclonal anti-HA antibody (Primary antibody) was added first followed by 30 minute treatment with vehicle (A) or 10μM isoproterenol (B). The cells were then fixed with 4% PFA and Alexafluor 640 conjugated secondary antibody was added for visualization. The cells were visualized using the Nikon eclipse 80i fluorescent microscope. (A) Cells treated with vehicle showed a uniform distribution around the cell. (B) Stimulation with isoproterenol localized the receptors to intracellular aggregates consistent with internalization into endosomes.

42 empty vector transfected cells at 140 ± 500 nM. In addition, the endogenous β2AR signal in the mock transfected cells is almost completely desensitized after 30 minutes while this was not the case with the transfected cells, in which the signal was persistent even after 30 minutes of stimulation (Compare empty vector transfected condition in both figure 3.3A and B).

Furthermore, the EC50 of the two transfected cells remained similar after 30 minutes of isoproterenol stimulation with EC50 values of 0.78±0.3 and 0.34±0.2 nM for the SS-HA-βlac- β2AR and HA-β2AR respectively, which also indicates a lack of desensitization in the transfected cells compared to the mock cells. In summary, the addition of the N-terminal βlac does not affect the signalling of the β2AR when compared to the HA- β2AR as measured using an EPAC BRET cAMP biosensor.

3.3 βlac-β2AR experiments

Having established that the presence of an N-terminal βlac does not alter β2AR pharmacology, the experimental conditions for measuring the internalization of SS-HA-βlac- β2AR using the βlac as a reporter were optimized. We used a concentration of 100μM of nitrocefin which is above the Km of the enzyme and has previously been used in the literature (Jones et al., 1982; De Meester et al., 1987; Bouillenne et al., 2000; Bebrone et al., 2001; Tan et al., 2003). Cells were seeded in a poly-d-lysine coated 96/48 well plate, and incubated for 24h at 37 ˚C. Initial studies showed that seeding stably expressing SS-HA-βlac-β2AR cells at a density of 100 000 cells/well led to a complete hydrolysis of nitrocefin within 5 minutes, which under our experimental conditions was considered to be too fast for the internalization studies. Therefore, cells were seeded at a lower density of 10 000 cells/well for future experiments using the SS-HA-βlac-β2AR stable cell line.

3.3.1 Comparison of Isoproterenol stimulated β2AR Internalization using the βlac and the ELISA Assays

Internalization of the β2AR by isoproterenol has been used by others in the validation of their assays for the quantification of surface expression (Hammer et al., 2007; Fisher et al., 2010; Wu et al., 2012b). Having optimized the conditions for measuring βlac activity for the SS- HA-βlac-β2AR stable cell line, the effects of isoproterenol on internalization of the SS-HA-βlac- β2AR were investigated in parallel to well established classical ELISA.

A 5 Minute Agonist Stimulation 0.20 50ng Blac-B2AR 0.15 50ng HA-B2AR

BRET) Empty Vector  0.10

0.05

0.00

-12 -10 -8 -6 -4 cAMP levels ( levels cAMP -0.05 log [Isoproterenol] M

B 30 Minute Agonist Stimulation 0.20 50ng Blac-B2AR 0.15 50ng HA-B2AR

BRET) Empty Vector  0.10

0.05

0.00

-12 -10 -8 -6 -4 cAMP levels ( levels cAMP -0.05 log [Isoproterenol] M

Figure 3.3: β2AR functional assay using the BRET cAMP EPAC biosensor. HEK293 cells stably expressing EPAC were transfected with the following: SS-HA-βlac-β2AR (red), HA- β2AR (blue), or empty vector (black). Cells were plated in white 96-well plates at 100,000 cells per well. Cells were washed once with PBS and coelenterazine H was added. After 5 minutes isoproterenol was added and the plate read once every 5 minutes on the Mithras luminometer.

(A) After 5 minutes isoproterenol stimulation the empty vector transfected cells had an EC50 of

120 nM. The SS-HA-βlac-β2AR and HA-β2AR EC50 were left shifted at 0.68±1.6 and 0.31 ±0.8 nM respectively. (B) After 30 minutes of isoproterenol stimulation the empty vector transfected cells had almost completely desensitized. The SS-HA-βlac-β2AR and HA-β2AR maintained their EC50 at 0.78±0.7 and 0.34±0.4 nM respectively. The empty vector transfected cells was a representative curve while the SS-HA-βlac-β2AR and HA-β2AR transfected cells were three independent experiments (n=3). All data are represented as mean of the background subtracted Rluc/YFP ratio ± S.E.M.

A B 120 120

100 100

80 80

Expression

% Cell %Surface Cell % Cell %Surface Cell 60 60

0 20 40 60 0 20 40 60 Time (min) Time (min)

C D 110 110 100 100 90 90 80 80

70 70 Expression

Expression 60 60

% Cell %Surface Cell % Cell %Surface Cell 50 50 40 40 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log [Isoproterenol] M log [Isoproterenol] M

Figure 3.4: Comparison of βlac and ELISA with the SS-HA-βlac-β2AR stable cell line. Cells for the βlac assay were seeded into 48 well plates at 10 000 cells/well and the ELISA was seeded at 100 000 cells/well. Time course of internalization of the SS-HA-βlac-β2AR was done on the βlac (A) and ELISA (B) where the cells were stimulated with a dose of 10μM isoproterenol once every five minutes. The half life values for βlac and ELISA internalization were 6.54±1.13 and 6.39±1.3 min respectively. The dose response for internalization for the SS-HA-βlac-β2AR was also done with the βlac assay (C) and ELISA (D). Cells were treated with a dose range of -4 -11 isoproterenol from 10 to 10 M and incubated for 30 minutes. An EC50 of 14.54±12.7 nM was obtained for internalization using the βlac assay; the ELISA yielded a linear relationship. All data are represented as mean of % vehicle treated ± S.E.M or three independent experiments (n=3).

120

100

% Cell Surface Expression Surface Cell % 60 -12 -10 -8 -6 -4 -2 log [Alprenolol] M B

120

100

% Cell Surface Expression %Surface Cell 60 -12 -10 -8 -6 -4 -2 log [Propranolol] M

Figure 3.5: Blocking of isoproterenol induced internalization with pre treatment of antagonists. SS-HA-βlac-β2AR cells were seeded in 48 well plates at 10 000 cells/well. Antagonists alprenolol (A) and propranolol (B) were added to the cells for 30 minutes from a dose range of 10-4 - 10-10 M. After antagonist incubation, cells were subsequently stimulated with a dose of 10μM isoproterenol for 30 minutes. Alprenolol and propranolol yielded IC50 values of 16.3±18.0 and 66.1±35.5 nM respectively. All data are represented as mean of % vehicle treated ± S.E.M of four independent experiments (n=4).

Parallel experiments were done to investigate the differences between the ELISA and βlac assay by looking at the time course and dose response of isoproterenol induced internalization (Figure 3.4). The time course of receptor internalization was measured after stimulating cells with a dose of 10μM isoproterenol in 5 minute intervals for 60 minutes. Both the βlac assay and ELISA yielded similar half-lives (6.54±1.13 and 6.39±1.3 minutes respectively) for internalization of β2AR (Figure 3.4A–B). In a second set of studies, the dose response of β2AR internalization was evaluated with a dose range of 10-4 to 10-11M of isoproterenol. After stimulating the cells for 30 minutes with various doses of isoproterenol an

EC50 of 14.54±12.7 nM was obtained for the βlac assay (Figure 3.4C). In comparison the ELISA yielded a linear relationship which does not allow us to calculate an EC50 value (Figure 3.4D). In summary, the parallel experiments showed the βlac assay was equally as quantitative as the ELISA in the time course experiments. However for the dose response of isoproterenol induced internalization, the βlac assay was able to yield an EC50 consistent to what has been reported in the literature (Hammer et al., 2007; Fisher et al., 2010) while the ELISA yielded a linear relationship for which an EC50 cannot be calculated.

3.3.2 Antagonist Blocking of Isoproterenol Induced Internalization of the β2AR

It is well established that agonist induced internalization can be blocked with the presence of antagonists. We investigated the ability of propranolol and alprenolol, two known β2AR antagonists, in blocking isoproterenol induced internalization of the SS-HA-βlac-β2AR. Cells were pre-treated for 30 minutes with the antagonists followed by the addition of 10μM isoproterenol. Both alprenolol and propranolol blocked isoproterenol mediated internalization in a dose dependent manner (Figure 3.5A and B). The IC50 values for alprenolol and propranolol for inhibition of isoproterenol induced internalization were 16.3±18.0 and 66.1±35.5 nM respectively.

3.3.3 Z’ Determination of the SS-HA-βlac-β2AR Internalization

In order to evaluate the robustness of the βlac assay for measuring β2AR internalization, the Z’ Factor of this specific assay was evaluated (Figure 3.5). The Z’ is a statistical measure regarding the quality of an assay where the Z’ takes into account both the signal to noise ratio as well as the variation between positive and negative controls. A Z’ value greater than 0.5 indicates the assay is suitable for screening purposes. These studies were carried out in a 96 well plate where the SS-

Z’=0.52

Figure 3.6: Z’ of the βlac assay using the SS-HA-βlac-β2AR stimulated with isoproterenol. SS-HA-βlac-β2AR cells were seeded into 96 well plates at 5000 cells/well. Evaluation of the Z’ of SS-HA-βlac-β2AR internalization was done after stimulation with isoproterenol. Cells were treated with either vehicle (blue) or 10µM isoproterenol (red) for 30 minutes and internalization was assessed using the using the βlac assay. The solid horizontal line represents the mean of the two conditions while the dotted liens represent 3 standard deviations away from the mean. The calculated value is Z’=0.52.

HA-βlac-β2AR stable cells were seeded at a density of 5000 cells/well. Cells were treated with either 10μM isoproterenol (Positive control) or vehicle (Negative control) for 30 minutes. Using the equation described in the methods sections (Section 2.14), the Z’ was determined to be 0.52, indicating the assay is robust enough to be suitable for screening purposes (Zhang et al., 1999b).

3.3.4 Pharmacological Chaperoning using β2AR Antagonists

In the last set of experiment with SS-HA-βlac-β2AR, the effects of alprenolol and propranolol as potential pharmacological chaperones of the β2AR were evaluated. It has been shown previously that both alprenolol and propranolol act as pharmacological chaperones for the β1AR (Kobayashi et al., 2009) and given that both alprenolol and propranolol are high affinity antagonists for β2AR as well as being lipophilic (logp = 2.9, ACD logP), it is proposed that these antagonists could potentially act as pharmacological chaperones for the β2AR as well. Using the βlac assay, the effects of an over-night treatment of alprenolol and propranolol on surface expression of β2AR were measured (Figure 3.7). Treatment with both antagonists (10μM) increased the surface expression of the SS-HA-βlac-β2AR. Alprenolol (Figure 3.7A) induced increase in surface expression could be measured starting at a dose of 1μM compared to propranolol (Figure 3.7B) which showed initial effects at a dose of 10μM. This result indicates that alprenolol has a greater potency than propranolol for increasing the surface expression, potentially through pharmacological chaperoning. Therefore the βlac assay has the ability to quantify increases in surface expression of β2AR after treatment with alprenolol and propranolol.

In summary the βlac assay was able to quantify the internalization profile of the SS-HA- βlac-β2AR. Specifically when the βlac assay is compared to the ELISA, both assays show similar results for the time course of β2AR internalization upon stimulation with isoproterenol.

The dose response for internalization yielded an EC50 for the βlac assay while the ELISA yielded a linear trend for internalization. Furthermore the βlac assay was also able to quantify antagonist blockade of agonist induced internalization. In addition the βlac assay was able to produce a Z’ = 0.52 for SS-HA-βlac-β2AR internalization; indicating this assay is suitable for screening purposes. Finally chronic/overnight treatment with the antagonists alprenolol and propranolol also increased the surface expression of the SS-HA-βlac-β2AR receptors consistent with the possibility of pharmacological chaperoning. Therefore these results indicate that βlac assay is a

A *** 150 ** 140 * 130

120

110

Expression % Cell %Surface Cell 100

0 -9 -8 -7 -6 -5 -4 log [Alprenolol] M

B ** 150 * 140

130

120

110

Expression % Cell %Surface Cell 100

0 -9 -8 -7 -6 -5 -4 log [Propranolol] M

Figure 3.7: Overnight incubation with alprenolol and propranolol increases surface expression of SS-HA-βlac-β2AR. SS-HA-βlac-β2ARcells were seeded into 48 well plates at 10 000 cells/well and were incubated overnight with antagonists (n=3). (A) Overnight incubation with alprenolol showed significant differences in surface expression were detected with doses of 1, 10, and 100 μM. (B) Overnight incubation with propranolol showed significant differences in surface expression were detected with doses of 10 and 100 μM. One-way ANOVA with bonferonni correct post-hoc were used to determine the differences between data sets * P<0.05, ** P<0.01, *** P<0.001. All data are represented as mean of % vehicle treated ± S.E.M.

50 suitable assay for measuring the effects of agonist induced internalization as well as the potential for pharmacological chaperoning.

3.4 Comparison of GBR1 Surface Expression using the βlac and the ELISA

GBR1 is a well characterized class C GPCR that requires the dimerization of the molecular chaperone GBR2 for its proper surface expression (see section 1.4.3). Therefore by using the SS-HA-βlac-GBR1 construct we investigated the ability of the βlac assays to quantify increases in surface expression due to molecular chaperoning. As with the internalization experiments, both the βlac and the ELISA experiments were done in parallel. The SS-HA-βlac- GBR1 stable cell line was transfected with an increasing amount of GBR2 and plated in a 48 well plate with 100,000 cells/well for both the βlac and ELISA assays. As shown in figure 3.8, the resulting increase in surface expression of the SS-HA-βlac-GBR1 after GBR2 transfection was quantitatively very similar between the ELISA and βlac assay. For both assays, the βlac and ELISA (Figure 3.8A and B) there was a statistically significant increase in surface expression of SS-HA-βlac-GBR1 at 0.22 μg of GBR2 transfection and above. In summary the βlac assay is equivalent to the ELISA in quantifying the increase in GBR1 surface expression due to molecular chaperoning from GBR2 co-expression.

A *** 3.0 ** 2.5

2.0

g g GBR2  1.5

1.0 Fold of 0 of Fold

0.5

g GBR2 g GBR2 g GBR2 g GBR2 g GBR2 g GBR2      

0.00 0.02 0.07 0.22 0.66 2.00

B *** ** 3.0 * 2.5

2.0

g g GBR2  1.5

1.0 Fold of 0 of Fold

0.5

g GBR2 g GBR2 g GBR2 g GBR2 g GBR2 g GBR2      

0.00 0.02 0.07 0.22 0.66 2.00

Figure 3.8: Comparison of βlac assay and ELISA Surface expression of GBR1 with GBR2 co-expression. SS-HA-βlac-GBR1 cells were transfected with differing amounts of GBR2 and seeded into 48 well plates at 100 000 cells/well (N=3). Quantification of the increase of GBR1 surface expression using the βlac assay (A) or ELISA (B) Both assays showed significant differences in surface expression when transfected with 0.22, 0.66, and 2.00 μg of GBR2. One- way ANOVA with bonferonni correct post-hoc were used to determine the differences between data sets * P<0.05, ** P<0.01, *** P<0.001. All data are represented as mean of % mock transfected cells ± S.E.M.

Chapter 4 Discussion

4.1 Summary of Key Findings

In the present study, we show that the βlac assay is a novel assay for the quantification of GPCRs. Our results show that the βlac assay is equally quantifiable as the classical approach, ELISA, when monitoring the time course of isoproterenol mediated internalization of the β2AR or the surface expression of GBR1. Furthermore, the βlac assay was able to quantify changes in β2AR surface expression after antagonist treatment hinting to the potential pharmacological chaperoning effects of these antagonists on β2AR.

4.2 Nitrocefin Permeability

One important aspect of the βlac assay that has not been addressed thus far is whether or not nitrocefin is cell impermeable. Although this has not been well established in the literature for eukaryotic cells, there are two lines of evidence that strongly suggest that nitrocefin is cell impermeable. First, the results obtained from βlac and ELISA experiments conducted in parallel are quantitatively and qualitatively similar, which would have not been the case if nitrocefin was cell permeable. Second, the cytoplasmic βlac-arrestin construct was utilized to experimentally measure cell permeability of nitrocefin (Pieter Beerepoot, Salahpour lab; Appendix Figure 1). Since βlac-arrestin is localized in the cytoplasm, the βlac is therefore not present at the plasma membrane, resulting in no nitrocefin hydrolysis if nitrocefin is cell impermeable. In this experiment there is little to no signal when nitrocefin is added to cells transfected with different amounts of βlac-arrestin. However when the cells are lysed using mechanical force and hypotonic shock, there is a large increase in signal upon addition of nitrocefin, indicating that nitrocefin did not cross the plasma membrane in the non-lysed, intact cells. Therefore based on the data presented in this experiment, nitrocefin is cell impermeable and is a suitable substrate for use in the quantification of surface expression of GPCRs.

4.3 Functional Experiments with the SS-HA-βlac-β2AR

The addition of a large N-terminal tag to GPCRs can potentially affect the function of the receptor. This is especially important when attempting to quantify effects of GPCR trafficking

(ie internalization) that is a direct result of GPCR signalling and ligand binding. It is therefore important to determine the effects that the βlac might have on the β2AR. The first experiment carried out was immunoflouresence of the SS-HA-βlac-β2AR, which showed a uniform distribution of the receptor at the cell surface when treated with vehicle, consistent with surface expression as shown in the literature(Kim et al., 2008). Upon stimulation with isoproterenol the receptor localizes to intracellular aggregates which is consistent with localization within endosomes as reported before (Cao et al., 2005).

To complement the immunofluoresence data, the signalling of the SS-HA-βlac-β2AR was compared in parallel with the HA-β2AR receptor. For these studies a BRET EPAC cAMP biosensor was used. This biosensor has been established as a robust method for assessing GPCR signalling through the Gαs pathway (Barak et al., 2008; Salahpour et al., 2012). The biosensor is formed by the addition of Renilla-luciferase (Rluc) and YFP to the N- and C-terminus of the EPAC protein. In its basal state the EPAC adopts a conformation such that the Rluc and YFP moieties are in close proximity resulting in high transfer of energy from the Rluc to the YFP and therefore high BRET levels. Upon binding of cAMP the EPAC adopts a conformation that moves the Rluc and YFP apart leading to a decrease in the BRET signal (Figure 4.1). Using this cAMP biosensor the signalling of the SS-HA-βlac-β2AR and HA-β2AR were assessed. Our results show that SS-HA-βlac-β2AR and HA-β2AR produce similar dose response curves and

EC50s with regards to isoproterenol induced cAMP signalling. It is important to note that it is possible that the HA tag could affect the function of the β2AR. However it has been shown previously that other small N-terminal epitopes (ie c-myc) on the β2AR did not affect binding of [I125]cyanpindolol compared to WT receptors(Angers et al., 2000). Lastly, it was observed that isoproterenol response in both the SS-HA-βlac-β2AR and HA-β2AR transfected cells did not desensitize after 30 minutes. It is possible that the heterologous expression of these receptors saturates the desensitization/internalization machinery of the HEK 293 cells. Indeed, this has been previously observed with the β3AR in other cell lines (Chaudhry and Granneman, 1994) and the AT1R (Violin et al., 2006a). Furthermore, studies with TAAR1 in HEK293 cells showed that transfection of β-arrestin 2 enables proper desensitization of this receptor (Barak et al., 2008). Therefore the cause of the lack of desensitization of recombinant β2AR in HEK 293 cells can be studied by either using different cell lines or by transfecting additional arrestins or GRKs into HEK 293 cells. In summary the SS-HA-βlac-β2AR and HA-β2AR dose response curves

cAMP

Substrate Rluc Substrate Rluc

475nm EPAC 475n EPA m YFP C YFP 525nm

Figure 4.1: Representative diagram for the BRET EPAC cAMP Biosensor. The EPAC biosensor contains an N- and C-terminal Rluc and YFP respectively. In its resting state the EPAC adopts a conformation such that the Rluc and YFP are within 100Å, allowomg for the transfer of energy from the Rluc to the YFP, resulting in a high BRET level (left panel). Upon binding of cAMP the EPAC adopts a conformation that moves the Rluc and YFP away decreasing the BRET (right panel).

have nearly identical EC50 values indicating that the addition of βlac at the N-terminus of the receptor does not affect its ability to signal through Gs. Additional experiments can be performed to further determine if the additional of the N-terminal βlac has any effect on β2AR function. Such experiments include radioligand binding of the receptor and determining the intracellular and extracellular pools of receptors for both the SS-HA-βlac-β2AR and HA-β2AR cells to determine if there is a difference in expression level.

4.4 SS-HA-βlac-β2AR Internalization

Our assay operates under the assumption that the enzyme reaction is operating under Michaelis-Menten kinetics. The substrate concentration used is above Km and presumably in excess of the enzyme, allowing for zero order kinetics. The assay is measured kinetically once every minute upon addition of substrate (nitrocefin) where the readout for the assay is the initial velocity that is measured as the initial slope of the kinetics of the reaction. In the future, additional characterization will be done on the enzyme kinetics in order to insure that the experimental conditions are appropriate.

In order to validate the βlac assay, the β2AR internalization profile of β2AR was evaluated using the βlac assay and ELISA in parallel. The isoproterenol induced internalization of the SS-HA-βlac-β2AR with the βlac assay yielded results that were consistent with the values in the literature. The dose response of isoproterenol induced β2AR internalization with βlac assay yielded EC50 values that were nearly identical to what has been reported with other surface expression assays (Hammer et al., 2007; Fisher et al., 2010; Takeda et al., 2012). In addition, the

EC50 of the βlac assay is similar to the EC50 (24.54 ± 12.7 nM) as measured by flow cytometry (Pieter Beerepoot, Salahpour lab; Appendix Figure 2). However when the dose response of internalization was done in parallel with an ELISA, the ELISA yielded a linear trend. The linear trend for the ELISA could potentially be due to an assay artefact. When looking at flow cytometry, a relatively similar assay protocol, the results yielded similar EC50 values to the βlac assay. The specific cause of this linear relationship remains elusive where additional experiments need to be done to determine the cause. One additional experiment to examine the cause of the linear trend in the ELISA would be to increase the dose range of isoproterenol in order to see if the response returns to a sigmoidal function.

The time course of isoproterenol induced internalization of the βlac-β2AR yielded very similar half-lives for the βlac and ELISA assays. Furthermore these values are in agreement with what has been previously reported for β2AR internalization (Moore et al., 1995). In addition these half-lives are also very similar to the half-life (4.52±0.74 minutes) that was obtained from flow cytometry (Pieter Beerepoot, Salahpour lab; Appendix Figure 3). However, in some other studies, using other surface expression assays, the half-lives of isoproterenol induced β2AR internalization is at least 2 fold higher than what we observed. These assays include the FAP biosensor, β-galactosidase complementation, and a endosomal BRET assay (Hammer et al., 2007; Fisher et al., 2010; Lan et al., 2011). It is important to note the experimental differences between these and the βlac assay. Two of the reported studies and assays quantify receptor internalization through the recruitment of the receptors into endosomes (Hammer et al., 2007; Lan et al., 2011) while the βlac assay and ELISA quantifies internalization through the absence of receptors on the cell surface. It is therefore possible that the time point for targeting a GPCR to an endosome is greater than that of internalization. However, a direct comparison between these assays and the βlac assay needs to be done in order to further examine why the half-life values are different for these different assays. All three assays have different maximum internalization values. While the ELISA and βlac assay have similar maximum internalization value (~40%), flow cytometry yielded ~60% internalization (see Appendix figure 2 and 3). It is probable that this difference is due to the fact that in flow cytometry, the antibody is done on cells in suspension while for ELISA and βlac the assays are carried out on adherent cells.

Lastly SS-HA-βlac-β2AR internalization by isoproterenol can be blocked through pre- treatment with antagonists alprenolol or propranolol in a dose dependent manner. The IC50 for alprenolol and propranolol were 16.3±18.0 and 66.1±35.5 nM respectively. The IC50 value for propranolol is similar to what has been reported in the literature (Hammer et al., 2007; Fisher et al., 2010), and it appears that our study is the first to report an IC50 value for alprenolol blocking of isoproterenol induced β2AR internalization. It is important to note that after 30 minutes of alprenolol treatment there was an increase in surface expression compared to vehicle treated cells (Figure 3.5). Since alprenolol is an inverse agonist for the β2AR (Varma et al., 1999) it is possible that the 30 minute treatment could have stabilized the receptors present on the plasma membrane. Interestingly however, this effect was not seen with propranolol treatment which is also an inverse agonist as well (Varma et al., 1999).

4.5 Pharmacological chaperoning

Using the βlac assay we observed that overnight treatment with either alprenolol or propranolol increased the surface expression of the SS-HA-βlac-β2AR. This experiment also showed that alprenolol increased the surface expression of the β2AR at a lower dose (1μM) compared to propranolol (10µM). The mechanism for why alprenolol is more potent than propranolol in increasing surface expression is not known. Although these doses for pharmacological chaperoning are rather high for the antagonists, the relationship between antagonist affinity and its ability to act as a pharmacological chaperone is not currently known. It is possible that the dose reported in this study is a function of both the affinity of the antagonist for the receptor as well as the ability of the compound to cross the plasma membrane. The affinity of these two antagonists are similar with Ki values of 0.4 and 1nM for propranolol and alprenolol respectively (Fraundorfer et al., 1994; Hoffmann et al., 2004). One potential explanation for this effect could be in relation to the off rate for these two antagonists where the relative off rate of alprenolol could be higher than propranolol; however this has not been shown in the literature.

In addition while this increase in surface expression is consistent with pharmacological chaperoning, additional experiments need to be done in order to determine the exact mechanism by which treatment with these antagonists leads to increased surface expression. Indeed, there could be multiple effects that increase surface expression of GPCR. For example, compounds could act by stabilizing the receptor in its inactive state decreasing constitutive activity and therefore stabilizing the receptor on the plasma membrane. Therefore, it is possible that inverse agonists may be more potent at stabilizing receptors at the cell surface than neutral antagonists. Indeed this mechanism of increasing surface expression through stabilization of the receptor at the plasma membrane has previously been reported for the histamine H2 (Smit et al., 1996) receptor as well as other constitutively active mutant receptors (Heinflink et al., 1995; Gether et al., 1997; Lee et al., 1997; Takeda et al., 2012). Indeed, since both alprenolol and propranolol act as inverse agonists on the β2AR they could potentially increase surface expression through this mechanism (Chidiac et al., 1994). This mechanism has been proposed for the in vivo action of nadolol, a hydrophilic β2AR antagonist (Walker et al., 2011). Second, the increase in surface expression could be due to pharmacological chaperoning since there is some percentage of GPCRs that are misfolded and subsequently degraded through the ER quality control mechanism

(Dong et al., 2007). A number of experiments could be performed in order to assess whether or not alprenolol and propranolol are pharmacological chaperones for the β2AR. First, the most established way of determining pharmacological chaperoning is the use of misfolding mutants that do not traffic to the plasma membrane (Morello et al., 2000; Kobayashi et al., 2009). If treatment of these mutants with alprenolol or propranolol rescued their surface expression, it would be good evidence of a pharmacological chaperoning effect of these compounds. Secondly incubation with a hydrophilic antagonist such as nadolol (logp = 0.56) should not increase the surface expression of the β2AR if the mechanism is exclusively through pharmacological chaperoning of nascent receptors within the ER (Morello et al., 2000).

In sum, we suggest that the effects of alprenolol and propranolol on surface expression of β2AR are possibly due to both the stabilization of the receptor at the plasma membrane as well as pharmacological chaperoning.

4.6 Z’ Factor of βlac-β2AR internalization

The Z’ is a statistical measure regarding the quality of an assay. The Z’ takes into account both the signal to noise ratio as well as the variation between positive and negative controls. A Z’ > 0.5 indicates an assay is suitable for screening purposes. Utilizing the internalization of the SS- HA-βlac-β2AR as a readout, a Z’=0.52 was obtained in a 96 well format. This Z’ indicates that the βlac assay, under these conditions, can be used to screen for compounds that induce internalization of the SS-HA-βlac-β2AR. It is important to note that we did not attempt to determine the Z’ for an ELISA using the SS-HA-βlac-β2AR cells. However we hypothesize that the Z’ for the ELISA would be lower than the βlac assay due to the variability as a result of the multiple wash steps.

Other studies have looked at the ability of other assays to yield a suitable Z’ for β2AR internalization. For the coiled coil tag assay, a Z’= 0.30 in a 96 well plate for β2AR internalization was reported, which indicates that this assay is not suitable for screening (Takeda et al., 2012). Next, the β-galactosidase complementation assay yielded a Z’=0.6 in a 384 well plate, a similar Z’ to the βlac assay (Hammer et al., 2007). The higher Z’ of the β-galactosidase assay is most likely due to automation of that assay which would decrease the variability of the assay and improve the Z’. Next, the FAP biosensor yielded a Z’= 0.72 in a 384 well plate (Wu et al., 2012b). The higher Z’ factor for this assay is most likely due to the increase in the maximum

59 measured internalization to 100% compared to 40% for the βlac assay. This increase in the maximal internalization could be a result of two factors. First the U937 cell line was used for the FAP biosensor experiments. Therefore, it is possible that the U937 cell line expressed a higher level of GRKs and/or arrestins leading to increased receptor phosphorylation and internalization. Indeed expression levels of arrestins and specific GRKs in HEK293 cells affect the internalization of the β2AR (Violin et al., 2006b, 2008). However the expression levels of arrestin and GRKs in U937 cells has not been studied. Secondly because the FAP biosensor is quantified using flow cytometry, it is possible that this method of measurement reports a higher rate of internalization than other methods. Indeed this is seen within our lab (Pieter Beerepoot, Salahpour lab; Appendix Figure 2 and 3) where using flow cytometry we have measured a greater percentage of internalized receptors compared to ELISA or βlac. Therefore the increase in Z’ for the FAP biosensor could potentially be due to these two factors listed above.

In order to improve the Z’ of the βlac assay for further miniaturization, several modifications to the system can be done. As listed above, transfecting arrestins and GRKs could increase the internalization of the receptors therefore increasing the signal/noise ratio. Furthermore, automation could be introduced to decrease the variability associated with manual pipetting. Lastly using different cell lines could increase the internalization of the receptors as seen with other assays (Hammer et al., 2007; Fisher et al., 2010; Wu et al., 2012b). These modifications could potentially further improve the Z’ of the βlac assay for internalization of β2AR.

Our future experiments include determining the Z’ for both molecular and pharmacological chaperoning using the βlac assay. We expect that these two measurement outputs should yield a higher Z’ than that of β2AR internalization. For molecular chaperoning, the negative non-transfected control should have little to no surface expression yielding a higher signal to background ratio for this assay in comparison to the β2AR internalization assay. Indeed our results show that transfecting 2μg of GBR2 into the SS-HA-βlac-GBR1 stable cell line yielded a 2.5 fold higher signal compared to mock transfected SS-HA-βlac-GBR1 cells. This is considerably higher than the 40% difference measured in internalization assays.

To determine the Z’ for pharmacological chaperoning, a misfolding GPCR mutant would be used. For example the vasopressin V2R del62-64 mutant has been previously shown to be

60 misfolded in the ER leading to a lack of surface expression of this receptor (Morello et al., 2000). Treatment of cells with SR 49059, a selective cell permeable antagonist of V2R, results in the rescue of surface expression of this mutant through a pharmacological chaperone effect. Therefore this mutant V2R receptor in conjunction with SR 49059 treatment could be used to determine the Z’ of the βlac assay for pharmacological chaperoning.

4.7 SS-HA- βlac-GBR1 Surface Expression by Co-expression with GBR2

In our last set of experiments we show that the βlac assay is able to quantify GBR1 cell surface expression in an equivalent manner to the ELISA. Although the ELISA appears to have a slightly greater magnitude of change compared to the βlac assay (Figure 3.8), the ELISA is also more variable as evidenced by the larger error bars.

It is important to note that studies within the Salahpour lab have shown that SS-HA- βlac- GBR1 receptor is able to signal in a similar manner to WT-GBR1 upon stimulation with baclofen, a GBR1 agonist (Pieter Beerepoot, Salahpour lab; Appendix Figure 4). This observation is in line with studies that have shown that the addition of the 30kDa SNAP tag to the N-terminus of GBR1 does not affect its function when compared to WT receptors (Maurel et al., 2008). It is important to note that interaction sites of GPCRs with molecular chaperones (in this case GBR2) reside in the transmembrane domain as well as the C-terminus of the GPCR (White et al., 1998; Wu et al., 2012a). Therefore tagging GPCRs on the N-terminus would seem an appropriate approach when investigating molecular chaperones.

4.8 Cost Analyses

The main advantages on the βlac assay are the simplicity and low cost relative to other assays that quantify surface expression of GPCRs (Table 1). When compared to an ELISA, the βlac assay is almost 10 fold less expensive in material agents alone. Furthermore, the βlac assay compares favorably to other surface expression assays such as the FAP biosensor and the SNAP/CLIP-tag assays due to the lack of commercial availability of the substrates for these assays. Indeed both the FAP and SNAP/CLIP-tag assays require chemical synthesis of their respective compounds before the assay can be carried out (Maurel et al., 2008; Szent-Gyorgyi et al., 2008). In summary, the βlac assay is a simple and cost effective assay for the quantification of surface expression of GPCRs.

Future Directions

Future experiments for βlac assay are split into two groups. First, we would continue to validate the assay and determine the Z’ factor for both pharmacological and molecular chaperoning as described section 4.6. Secondly once the Z’ factors are found we would like to screen for either molecular or pharmacological chaperones for receptors that do not express on the plasma membrane (ie. TAAR1).

Conclusion

In this study we have demonstrated that the creation of an N-terminal βlac-GPCR fusion protein allows for the quantification of surface expression. Through the use of the SS-HA-βlac- β2AR and SS-HA-βlac-GBR1, the βlac assay has been validated for use in quantifying internalization (dose response, time course, and antagonist blockade), increase in surface expression due to molecular chaperoning and potentially pharmacological chaperoning. When comparing the βlac assay with an ELISA, the assays performed very similarly. The main advantages of the βlac assay are its low cost and simplicity compared to ELISA or other traditional assays (Table 1). Given the advantages in cost, simplicity and most importantly robustness, the βlac assay is now the standard assay for quantification of surface expression of GPCRs within the Salahpour lab, where ELISAs are no longer performed. Therefore to conclude this study, the βlac assay is a novel assay that can be readily adopted in any laboratory due to its fast, robust, reproducible and cost effective qualities compared to all other assays currently available for quantifying surface expression.

Table 1: Comparison of time and cost of ELISA and βlac assays steps ELISA βlac

1 Wash (1 time) Wash (1 time)

2 Block Add nitrocefin

3 Add 1° antibody Read (Abs 486nm)

4 Wash (3 times)

5 Fix (4% PFA)

6 Wash (3 times)

7 Block

8 Add 2° antibody

9 Wash (3 times)

10 Add substrate

11 Stop reaction

12 Read (Abs 496nm) time 4- 6 hours 15-60 minutes cost 30-50 cents/well 4 cents/well

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Appendices

50 ** Mock Transfected 40 0.5 mg blac-bArrestin 1.0 mg blac-bArrestin 30 2.0 mg blac-bArrestin

10 Fold Increase in Signal in Increase Fold 0

O O 2 2

- ddH + ddH

Appendix Figure 1: Cell permeability of the βlac substrate nitrocefin. A rat β-arrestin2-βlac construct was created by replacing the YFP in the β-arrestin-YFP construct with βlac (Angers et al., 2000). Cells were transfected with 0, 0.5, 1, and 2 μg of βarrestin- βlac DNA, and were plated in a 48-well plate 24 hours post-transfection. At 48 hours post-transfection, cells were washed and incubated for 15 minutes in either PBS or ddH20. After incubation, cell solutions in the ddH20 treated wells were mechanically lysed by pipetting up and down. PBS was aspirated from the PBS-treated wells and nitrocefin in PBS was added. To the ddH2O treated wells, a solutioin of 2X nitrocefin in 2X PBS was then added, and nitrocefin hydrolysis was monitored. Unlysed cells showed no increase in signal compared to mock transfected cells, regardless of the amount of βarrestin-βlac DNA that was transfected. Cell lysis resulted in increases in signal with increasing amounts of βarrestin-βlac DNA reaching 33±6.8.fold compared to 2±0.9 fold for lysed mock cells. Two-way ANOVA with Bonferroni correct t-test post-hoc were used to determine differences between data sets ** P<0.01. All data represented as mean fold increase in signal from mock transfected HEK 293 cells ± S.E.M, n=3.

100

Expression 60 % Cell %Surface Cell

40 -12 -10 -8 -6 -4 -2 log [Isoproterenol] M

Appendix Figure 2: Dose response of isoproterenol mediated internalization quantified with flow cytometry. Cells were seeded into 6 well plates at 4 000 000 cells/well. The cells were stimulated with isoproterenol in a dose range of 10-4 0 10-11 M. Immediately after completion of drug treatment cells were put on ice and washed with cold PBS. The cells were lifted off by incubation for 10 min with PBS 0.02%EDTA.The EDTA was neutralized by adding cell culture media (DMEM 10% FBS). The suspensions were then spun down in a centrifuge with a swinging bucket rotor at 1500 rpm for 5 minutes and washed twice with PBS 2% FBS. Cells were spun down and resuspended in PBS 2%FBS with 1:250 primary antibody and incubated for 30 minutes. Subsequently, cells were spun down and washed twice in PBS 2%FBS. After centrifugation cells were incubated in 1:100 Alexa Fluor 647 Donkey anti-mouse IgG antibody (Invitrogen) in 100 μL PBS 2% FBS for 15 minutes. After 2 more PBS 2% FBS washes, the cells were fixed in PBS 2% PFA for 30 minutes, spun down, and resuspended in PBS 2%FBS. Subsequently, the cell suspensions were strained through cell strainer caps (BD Falcon) into round bottom tubes. Fluorescence was then acquired on a BD LSR Fortessa flow cytometer with an excitation wavelength of 640 nm, and a 670/14 bandpass filter, collecting 10000 events per sample. All steps were performed on ice and samples were protected from light after addition of secondary antibody. The EC50 for SS-HA-βlac-β2AR internalization was 24.54

± 12.66 nM. All data are represented as mean of % vehicle treated ± S.E.M or three independent experiments (n=3).

100

Expression 60 % Cell %Surface Cell

40 0 20 40 60 Time (min)

Appendix Figure 3: Time course of isoproterenol mediated internalization quantified with flow cytometry. Cells were seeded into 6 well plates at 4 000 000 cells/well. The cells were stimulated with isoproterenol at a dose of 10μM at time points of 0, 5, 10, 15, 30, and 50 minutes. Immediately after completion of drug treatment cells were put on ice and washed with cold PBS. The cells were lifted off by incubation for 10 min with PBS 0.02%EDTA.The EDTA was neutralized by adding cell culture media (DMEM 10% FBS). The suspensions were then spun down in a centrifuge with a swinging bucket rotor at 1500 rpm for 5 minutes and washed twice with PBS 2% FBS. Cells were spun down and resuspended in PBS 2%FBS with 1:250 primary antibody and incubated for 30 minutes. Subsequently, cells were spun down and washed twice in PBS 2%FBS. After centrifugation cells were incubated in 1:100 Alexa Fluor 647 Donkey anti-mouse IgG antibody (Invitrogen) in 100 μL PBS 2% FBS for 15 minutes. After 2 more PBS 2% FBS washes, the cells were fixed in PBS 2% PFA for 30 minutes, spun down, and resuspended in PBS 2%FBS. Subsequently, the cell suspensions were strained through cell strainer caps (BD Falcon) into round bottom tubes. Fluorescence was then acquired on a BD LSR Fortessa flow cytometer with an excitation wavelength of 640 nm, and a 670/14 bandpass filter, collecting 10000 events per sample. All steps were performed on ice and samples were protected from light after addition of secondary antibody. The half life of internalization for SS-

HA-βlac-β2AR internalization was 4.52±0.74 min. All data are represented as mean of % vehicle treated ± S.E.M or three independent experiments (n=3).

Appendix Figure 4: Functionality of SS-HA-βlac-GBR1 using the BRET EPAC cAMP biosensor. HEK293 cells stably expressing EPAC were transfected with the following: WT- GBR1 or SS-HA-βlac-GBR1 with 0.5-5μg GBR2. Cells were plated in white 96-well plates at 100,000 cells per well. Cells were washed once with PBS and coelenterazine H was added. After 5 minutes isoproterenol was added and the plate read once every 5 minutes on the Mithras luminometer. Treatment with forskolin results in formation of cAMP and therefore a change in BRET. This forskolin signal is partially blocked by -4M but not –9M baclofen treatment in the wildtype and βlac tagged GBR1. t-tests were performed to compare the effect of -4M baclofen to forskolin only treated cells n=4, * P<0.05, ** P<0.01