D 1610 OULU 2021 D 1610

UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA

DMEDICA Orvokki Mattila Orvokki Mattila Orvokki University Lecturer Tuomo Glumoff PROTEOLYTIC PROCESSING University Lecturer Santeri Palviainen OF G -COUPLED

Postdoctoral researcher Jani Peräntie RECEPTOR 37 BY ADAM10 AND FURIN University Lecturer Anne Tuomisto

University Lecturer Veli-Matti Ulvinen

Planning Director Pertti Tikkanen

Professor Jari Juga

Associate Professor (tenure) Anu Soikkeli

University Lecturer Santeri Palviainen UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE, Publications Editor Kirsti Nurkkala MEDICAL RESEARCH CENTER OULU

ISBN 978-952-62-2901-0 (Paperback) ISBN 978-952-62-2902-7 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

ACTA UNIVERSITATIS OULUENSIS D Medica 1610

ORVOKKI MATTILA

PROTEOLYTIC PROCESSING OF G PROTEIN-COUPLED RECEPTOR 37 BY ADAM10 AND FURIN

Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Auditorium F202 of the Faculty of Medicine (Aapistie 5 B), on 23 April 2021, at 12 noon

UNIVERSITY OF OULU, OULU 2021 Copyright © 2021 Acta Univ. Oul. D 1610, 2021

Supervised by Docent Ulla Petäjä-Repo

Reviewed by Docent Henri Xhaard Docent Annakaisa Haapasalo

Opponent Professor Pertti Panula

ISBN 978-952-62-2901-0 (Paperback) ISBN 978-952-62-2902-7 (PDF)

ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online)

Cover Design Raimo Ahonen

PUNAMUSTA TAMPERE 2021 Mattila, Orvokki, Proteolytic processing of G protein-coupled receptor 37 by ADAM10 and furin. University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Medical Research Center Oulu Acta Univ. Oul. D 1610, 2021 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract The G protein-coupled receptor 37 (GPR37) is a member of a large superfamily of integral membrane involved in signal transduction across the cellular plasma membrane. G protein-coupled receptors participate in many vital physiological processes and form a major class of drug targets. GPR37 is abundantly expressed in the brain and has been linked to dopaminergic and adenosinergic signaling, as well as oligodendrocyte differentiation and myelination. Moreover, the receptor has been implicated in certain brain pathologies, the most notable of which is Parkinson’s disease. However, the endogenous ligand of GPR37 has not been identified, and thus, the potential of GPR37 as a therapeutic target remains unexplored. The mechanisms that regulate GPR37 in cells are poorly characterized. Therefore, this study aimed to shed light on the biosynthesis, maturation and processing of GPR37 by using a heterologous expression system and various biochemical and cell biological methods. In particular, the limited proteolysis of the receptor N-terminus and the enzymes involved in that process were assessed. The results show that GPR37 matures rapidly and efficiently. However, the N-terminal domain of the receptor is subject to proteolytic processing at Arg54↓Asp55 and Glu167↓Gln168. The cleavage at the Glu167↓Gln168 site is very efficient and occurs constitutively. The N-terminal ectodomain is shed from cells and the remaining truncated receptor represents the predominant GPR37 species at the cell surface. The cleavage at the Arg54↓Asp55 site is less efficient and takes place in the trans-Golgi network during receptor trafficking to the cell surface. Characterization of the cleaving enzymes revealed that GPR37 is processed by a proprotein convertase furin at the Arg54↓Asp55 site and by a disintegrin and metalloprotease 10 (ADAM10) at the Glu167↓Gln168 site. These findings were confirmed using various complementary methods and cultured cell lines of diverse tissue origin. Importantly, the ADAM10-mediated cleavage was also found to occur in a more physiological context of mouse brain tissue. This study reveals a novel processing mechanism for GPR37. N-terminal cleavage is likely an important element for receptor regulation, and the results pave way for strategies to exploit GPR37 as a therapeutic target.

Keywords: ADAM proteins, cell surface receptors, furin, G protein-coupled receptors, GPR37, limited proteolysis, membrane proteins, metalloproteases, Parkinson’s disease, post-translational protein processing, proprotein convertase

Mattila, Orvokki, G-proteiiniin kytketyn reseptori 37:n ADAM10- ja furiinivälitteinen proteolyyttinen prosessointi. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Medical Research Center Oulu Acta Univ. Oul. D 1610, 2021 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä G-proteiiniin kytketty reseptori 37 (GPR37) kuuluu laajaan solun ulkopuolelta solun sisälle sig- naaleja välittävään kalvoproteiinien perheeseen. G-proteiiniin kytketyt reseptorit osallistuvat useisiin fysiologisesti merkittäviin toimintoihin, ja suuri osa markkinoilla olevista lääkkeistä vai- kuttaakin solun toimintaan niiden kautta. GPR37 on aivoissa voimakkaasti ilmentyvä reseptori, jonka on osoitettu liittyvän muun muassa dopaminergiseen ja adenosinergiseen signalointiin sekä oligodendrosyyttien erilaistumiseen ja myelinisaatioon. Lisäksi GPR37 on yhdistetty eräi- siin neurologisiin sairauksiin, joista merkittävin on Parkinsonin tauti. GPR37:n aktivoivaa ligan- dia ei kuitenkaan tunneta, ja tästä syystä reseptorin käyttöä lääkekohteena ei ole pystytty hyö- dyntämään. GPR37:n säätelyyn liittyvät mekanismit ovat huonosti tunnettuja. Siksi tässä työssä tutkittiin GPR37:n biosynteesiä ja prosessointia käyttäen heterologisia solumalleja sekä biokemiallisia ja solubiologisia menetelmiä. Työssä tutkittiin etenkin reseptorin aminoterminaalista proteolyyttis- tä prosessointia ja siihen liittyviä entsyymejä. GPR37:n havaittiin maturoituvan nopeasti ja tehokkaasti. Reseptorin solukalvon ulkopuolel- la sijaitseva aminoterminaalinen osa katkaistaan kuitenkin proteolyyttisesti kohdista Arg54↓Asp55 ja Glu167↓Gln168. Katkaisu kohdasta Glu167↓Gln168 tapahtuu tehokkaasti ilman ulkopuolista stimulaatiota. Aminoterminaalinen osa vapautuu soluista, ja jäljelle jäävä katkaistu reseptori edustaa solun pinnalla yleisimmin esiintyvää reseptorimuotoa. Katkaisu koh- dasta Arg54↓Asp55 on tehokkuudeltaan heikompi ja tapahtuu trans-Golgissa reseptorin kuljetus- reitillä solun pinnalle. Reseptorin katkaisuun liittyvien entsyymien karakterisointi osoitti, että proproteiinikonver- taasi furiini katkaisee GPR37:n kohdasta Arg54↓Asp55 ja metalloproteaasi ADAM10 kohdasta Glu167↓Gln168. Nämä havainnot vahvistettiin usealla toisiaan tukevalla menetelmällä käyttä- mällä erilaisia kudosalkuperiä olevia solulinjoja. Lisäksi ADAM10-välitteisen katkaisun havait- tiin tapahtuvan myös hiiren aivokudoksessa, mikä vahvistaa löydöksen fysiologisen merkityk- sen. Tässä työssä tunnistettiin uusi mekanismi, jolla GPR37:ää muokataan soluissa. Aminotermi- naalinen katkaisu on todennäköisesti tärkeä GPR37:ää säätelevä mekanismi, ja työn tulokset aut- tavat selvittämään reseptorin terapeuttista potentiaalia.

Asiasanat: ADAM-proteiinit, furiini, G-proteiiniin kytketyt reseptorit, GPR37, kalvoproteiinit, metalloproteaasit, Parkinsonin tauti, proproteiinikonvertaasi, rajoitettu proteolyysi, solun pintareseptorit, translaation jälkeinen proteiinikäsittely

To my family

8 Acknowledgements

This work was carried out at the Cancer and Translational Medicine Research Unit (2013-2016) and the Research Unit of Biomedicine (2016-2020) at the Faculty of Medicine, University of Oulu. Our research group, led by Docent Ulla Petäjä-Repo, was a member of the Medical Research Center Oulu during the time of the research. This study was financially supported by personal grants and salary from the University of Oulu, Magnus Ehrnrooth Foundation, Finnish Concordia Fund, Finnish Parkinson Foundation and Orion Research Foundation, by travel grants from the University of Oulu Graduate School, Oskar Öflunds Stiftelse and the Federation of European Biochemical Societies and by research group funding from the Academy of Finland, Medical Research Center Oulu and Magnus Ehrnrooth Foundation. I would like to express my sincere gratitude to my supervisor Docent Ulla Petäjä-Repo for her guidance during this thesis project. I am grateful to her for giving me the opportunity to work in such an interesting and challenging research project and for sharing her excellent knowledge of GPCRs. She has the ability to spot the important details that other people easily miss. Her thoughtful advice and expertise in the field of GPCRs made this thesis possible. I am grateful to the head of the Cancer and Translational Medicine Research Unit, Juha Tuukkanen, and the head of the Research Unit of Biomedicine, Jukka Hakkola for providing me the opportunity to work in an inspiring environment. I also thank Professors Petri Lehenkari and Risto Kerkelä for their supportive attitude towards my research. I thank Professor Pertti Panula for his willingness to participate in my doctoral defense as my opponent. I also wish to thank my pre-examiners Docents Henri Xhaard and Annakaisa Haapasalo for their valuable comments on this thesis. I am grateful to Deborah Kaska and Jukka Käräjäoja for the revision of the language of the thesis. I want to thank all the past and present members of the GPCR research group, especially Jarkko Lackman, Hanna Tuhkanen, Anja Konzack, Hamayun Khan, Mervi Kreus and Olli-Pekka Tapio with whom I had chance to share my work for several years. Jarkko and Hanna have helped me through the ups and downs of research life and made this journey more enjoyable. Hanna is a genuinely warm- hearted person who always has patience and time to help others. Jarkko has supported me in numerous ways in the lab, in the office and outside of work. I owe my deepest gratitude to him for sincere friendship and for his ability to lower my

9 stress levels. I am grateful to Anja for her support and help with the final experiments needed to finish this project. I also wish to thank Jussi Tuusa for his insightful ideas and for the original findings that made the beginning of my Ph.D. project easier for me. I am also grateful to Miia Vierimaa and Tuula Taskinen for their patience and skillful technical assistance. I feel a deep sense of gratitude to my follow-up group members Docents Alexander Kastaniotis, Anthony Heape and Johanna Magga for their support and help during difficult times. I wish to thank Helena Miettunen for encouragement during writing process of this thesis. I also want to express my warmest thanks to Marja, Mahmoud and Heba, who shared the same office with me, and all the scientists and technical staff at “Farmis” and the former department of Anatomy for their continuous support. I wish to thank Professor Michel Bouvier, Dr. Katrine Schjoldager and Professor Francisco Ciruela for giving me an opportunity to join their research groups in Montreal, Copenhagen and Barcelona during my research visits. I am also grateful to the members of their research teams for providing me with friendly and supportive working environment. Especially, I want to thank Francisco and his research group members Xavier Morató and Josep Argerich for their contribution to my research project and for all the insightful discussions about science. I also wish to thank Professor Paul Saftig for collaboration. I owe my deepest gratitude to my family and friends for their support and encouragement during this long thesis project. My mother Ulla, with her strength and perseverance, has always inspired me to try my best in everything I do. I want to thank her for her endless love and support. I am grateful to my father Seppo, my brother Riku and my sisters Sini, Heli and Outi for their love and encouragement. Anna, my dear friend since childhood, has shared with me the struggles of the academic life. I want to thank her for her support and for the countless conversations about research and life in general. I also wish to pay tribute to my cats Maddy and Riku (and Jori for the first five years of my Ph.D. project) for bringing joy into my life after long days in the lab and for keeping me company while I was writing this thesis.

Oulu, March 2021 Orvokki Mattila

10 Abbreviations

A2AR ADAM a disintegrin and metalloprotease AR-JP autosomal recessive juvenile parkinsonism BSA bovine serum albumin C-terminus carboxyl-terminus

D2R dopamine D2 receptor decCMK Decanoyl-Arg-Val-Lys-Arg-chloromethylketone DMEM Dulbecco’s modified Eagle medium ECL extracellular loop EDTA ethylenediaminetetraacetic acid Endo H endo-β-N-acetylglucosaminidase H ER endoplasmic reticulum ERAD endoplasmic reticulum-associated degradation ERGIC endoplasmic reticulum-Golgi intermediate compartment ERK1/2 extracellular signal-regulated kinase 1/2

ETBR FACS fluorescence-activated cell sorting FBS fetal bovine serum FC flow cytometry GABA gamma-aminobutyric acid GAIN G protein-coupled receptor autoproteolysis-inducing GDP guanosine diphosphate GPCR G protein-coupled receptor GPR37 G protein-coupled receptor 37 GPR37L1 G protein-coupled receptor 37 like 1 G protein guanine nucleotide-binding protein GRAFS Glutamate, , Adhesion, /Taste2, Secretin classification system GTP guanosine triphosphate h human HA hemagglutinin HEK human embryonic kidney HRP horse radish peroxidase ICL intracellular loop IF immunofluorescence

11 IUPHAR International Union of Pharmacology m mouse MAG myelin-associated glycoprotein MEF mouse embryonic fibroblast MMP matrix metalloprotease NC- IUPHAR International Union of Pharmacology, Committee on Receptor Nomenclature and Classification NEM N-ethylmaleimide NHS N-hydroxysuccinimide N-terminus amino-terminus PAR protease-activated receptor PBS phosphate-buffered saline PCR polymerase chain reaction PDZ postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 PEI polyethylenimine PINK1 PTEN-induced kinase 1 PNGase F peptide N-glycosidase F PVDF polyvinylidene fluoride SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis siRNA small interfering ribonucleic acid TBS Tris-buffered saline TGN trans-Golgi network TM transmembrane TSHR thyroid-stimulating hormone receptor WB western blotting WT wild-type

12 Original publications

This thesis is based on the following publications, which are referred to throughout the text by their Roman numerals:

I Mattila, S. O., Tuusa, J. T., & Petäjä-Repo, U. E. (2016) The Parkinson’s disease- associated receptor GPR37 undergoes metalloprotease-mediated N-terminal cleavage and ectodomain shedding. Journal of Cell Science, 129(7), 1366-1377. doi: 10.1242/jcs.176115 II Mattila, S. O., Tuhkanen, H. E., Lackman, J. J., Konzack, A., Morató, X., Argerich, J., Saftig, P., Ciruela, F., & Petäjä-Repo, U. E. (2020) GPR37 is processed in the N- terminal ectodomain by ADAM10 and furin. The FASEB Journal, Under revision

Additional unpublished data are also presented.

13

14 Contents

Abstract Tiivistelmä Acknowledgements 9 Abbreviations 11 Original publications 13 Contents 15 1 Introduction 17 2 Review of the literature 19 2.1 G protein-coupled receptors (GPCRs) ...... 19 2.1.1 Classification ...... 19 2.1.2 Structure ...... 21 2.1.3 Biosynthesis and trafficking in cells ...... 26 2.1.4 Signaling ...... 33 2.1.5 Orphan GPCRs ...... 41 2.2 GPR37 ...... 43 2.2.1 Phylogenetic classification ...... 43 2.2.2 Structural features ...... 45 2.2.3 Function ...... 49 2.3 Proteolytic processing ...... 53 2.3.1 Proprotein convertases ...... 54 2.3.2 ADAM family metalloproteases ...... 55 2.3.3 Ectodomain shedding ...... 58 2.3.4 Proteolytic processing of GPCRs ...... 58 3 Aims 61 4 Materials and methods 63 4.1 Receptor expression ...... 64 4.1.1 Animals ...... 64 4.1.2 DNA constructs ...... 64 4.1.3 Cell lines ...... 66 4.2 Cell culture methods ...... 68 4.2.1 Transient transfections ...... 68 4.2.2 Small interfering RNA-mediated silencing ...... 68 4.2.3 Cell surface biotinylation ...... 68 4.2.4 Metabolic pulse-chase labeling ...... 69 4.3 Protein extraction and purification ...... 69

15 4.3.1 Preparation of whole cell extracts ...... 69 4.3.2 Preparation and solubilization of cellular membranes ...... 70 4.3.3 Purification of solubilized proteins ...... 71 4.3.4 Enzymatic deglycosylation ...... 72 4.3.5 Purification of the N-terminal receptor fragment from the cell culture medium ...... 72 4.4 Protein detection ...... 73 4.4.1 SDS-PAGE and western blotting ...... 73 4.4.2 Flow cytometry ...... 74 4.4.3 Confocal microscopy ...... 75 4.4.4 N-terminal sequencing ...... 76 4.5 Data analysis ...... 77 5 Results 79 5.1 Biosynthesis and expression of GPR37 in mammalian cells ...... 79 5.1.1 Characterization of the expressed GPR37 species (I) ...... 79 5.1.2 Maturation of GPR37 (I, II) ...... 80 5.1.3 Characterization of transiently expressed receptors (I, II) ...... 81 5.2 Processing of GPR37 at the major cleavage site ...... 81 5.2.1 Cellular localization of GPR37 species (I, II)...... 82 5.2.2 Metalloprotease cleavage of GPR37 (I, II) ...... 82 5.2.3 Ectodomain shedding and cleavage site identification (I) ...... 83 5.3 Identification of the enzymes responsible for GPR37 cleavage ...... 84 5.3.1 ADAM10 cleavage between Glu167 and Gln168 (II) ...... 85 5.3.2 Furin cleavage between Arg54 and Asp55 (II) ...... 86 5.4 ADAM10 as a physiologically relevant GPR37 cleaving enzyme (II) ...... 88 6 Discussion 91 6.1 N-terminal cleavage of GPR37 ...... 91 6.2 Enzymes responsible for GPR37 cleavage ...... 93 6.3 Functional significance of GPR37 cleavage ...... 96 7 Conclusions 99 List of references 101 Original publications 127

16 1 Introduction

G-protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that mediate signals from the extracellular environment to the cell interior. They share a common architecture of seven transmembrane (TM) regions connected by extracellular loops (ECLs) and intracellular loops (ICLs). GPCRs can respond to a wide variety of extracellular stimuli ranging from proteins, hormones and neurotransmitters to ions and photons. Messages from these extracellular activators, collectively called ligands, are translated into intracellular signaling cascades via interactions with cytoplasmic guanine nucleotide-binding proteins (G proteins) and other signaling components. Owing to their broad expression pattern in distinct tissue types and large number of interacting partners, GPCRs are involved in virtually all aspects of human physiology (Wacker, Stevens, & Roth, 2017; Wootten, Christopoulos, Marti-Solano, Babu, & Sexton, 2018). The contains more than 800 GPCRs (Fredriksson, Lagerstrom, Lundin, & Schioth, 2003). They are attractive targets for pharmaceutical intervention, and more than one third of currently available drugs act through these receptors (Hauser et al., 2018). However, approximately 100 GPCRs, called orphan GPCRs, still remain without a known activating ligand (Laschet, Dupuis, & Hanson, 2018). Thus, their potential as drug targets remains to be resolved. The G protein- coupled receptor 37 (GPR37) is one of these relatively poorly characterized GPCRs without a verified endogenous ligand. The receptor is predominantly expressed in the brain and has been linked to dopaminergic and adenosinergic signaling pathways (Imai et al., 2007; Marazziti et al., 2007; Morato et al., 2019; Morato et al., 2017). In line with these findings, GPR37 has been implicated in Parkinson’s disease (Imai et al., 2001; Murakami et al., 2004) and certain other brain disorders (Fujita-Jimbo et al., 2012; Tomita et al., 2013). More recent research has extended the potential physiological role of the receptor to oligodendrocyte differentiation and myelination (B. M. Smith et al., 2017; Yang, Vainshtein, Maik-Rachline, & Peles, 2016). Therefore, GPR37 could be a promising target for pharmaceutical treatment of neurological diseases. Although studies with GPR37 transgenic and knock-out mice have provided some insights into physiological and pathophysiological roles of the receptor (Imai et al., 2007; Marazziti et al., 2004; B. M. Smith et al., 2017; X. Zhang et al., 2020), the basic biochemical mechanisms that regulate GPR37 in cells are poorly understood. Therefore, this study was conducted to elucidate the biosynthesis, maturation and processing of GPR37. An understanding of these aspects of GPR37

17 biology is imperative to begin to unravel the mechanisms involved in receptor activation and functional regulation.

18 2 Review of the literature

2.1 G protein-coupled receptors (GPCRs)

2.1.1 Classification

The first GPCR cDNA sequences were published in the 1980s, and as a consequence of intense cloning of new GPCRs, it soon became obvious that these receptors form a large and structurally diverse group of membrane proteins (Hargrave et al., 1983; Lagerstrom & Schioth, 2008; Nathans & Hogness, 1983). A few classification systems, which aim to divide GPCRs into smaller subfamilies, have emerged since the early 1990s. These classifications are typically established on the basis of homology, structural similarities or ligand properties. One of the earliest GPCR classifications was presented by Kolakowski who divided receptors into families A-F based on (Kolakowski, 1994). An alternative system was introduced in 1999 when Bockaert and Pin described a classification that groups GPCRs into families 1-5 according to their structure and ligand binding properties (Bockaert & Pin, 1999). More recently, a phylogenetic analysis of human GPCRs led to creation of the GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, Secretin) classification, the name derived from the initial letters of the prototypical representatives of each receptor subfamily. However, more recent evidence suggests that Taste2 receptors form a separate group from the Frizzled family as these receptors appear to originate from Rhodopsin family receptors (Nordstrom, Sallman Almen, Edstam, Fredriksson, & Schioth, 2011). The A-F system is the most commonly used classification in the literature, although the GRAFS system is also relatively popular. While there are some differences between the two classification systems, they are largely overlapping (Table 1). However, families D-E in the A-F system represent invertebrate receptor families with no known human orthologs, and thus, are not included in the GRAFS classification (Alexander et al., 2019). The International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) uses nomenclature that is based on these two classifications. The system divides human GPCRs into five families: Classes A-C, Adhesion and Frizzled (Table 1). Additionally, there are certain seven transmembrane-spanning proteins that do not belong to any of the identified receptor families, and therefore, form a separate group called Other 7TM proteins

19 (Alexander et al., 2019; Fredriksson et al., 2003). GPCRs can be additionally divided into sensory and non-sensory receptors based on their functional properties. Moreover, orphan receptors, which are GPCRs without a known ligand, are often mentioned separately from the receptors with identified ligands (Alexander et al., 2019).

Table 1. Classification of human GPCRs.

Alternative classification Number of receptorsa NC-IUPHAR GRAFS A-F Total Orphan Sensory Class Ab Rhodopsin A 719 87 435 Class B Secretin B 15 0 0 Class C Glutamate C 22 8 3 Adhesion Adhesion B 33 26 0 Frizzled Frizzled F 11 0 0 Other Other - 5 5 0 a The numbers of receptors as listed in the Concise guide to pharmacology 2019/20 (Alexander et al., 2019). b NC-IUPHAR classification includes Taste2 receptors in the Class A family. Abbreviations: NC- IUPHAR, International Union of Basic and Clinical Pharmacology, Committee on Receptor Nomenclature and Classification.

The Class A/ Rhodopsin family is the largest GPCR subfamily with a total number of over 700 receptors in humans (Alexander et al., 2019; Fredriksson et al., 2003). In the GRAFS classification, the family is subdivided into four groups termed α, β, γ and δ, and these groups are further divided into 13 subbranches of closely related GPCRs (Fredriksson et al., 2003). Members of this GPCR family are structurally relatively diverse and can bind a wide range of ligands such as purines, amines and peptides. Thus, they can be alternatively subdivided into eleven groups based on the types of their endogenous ligands (Munk et al., 2016; Munk et al., 2019). The Class B/ Secretin family is a small subgroup of GPCRs that bind peptide hormone ligands via an extracellular hormone-binding domain (Lagerstrom & Schioth, 2008). These receptors are further clustered into five subgroups based on their physiological functions (de Graaf et al., 2017). Although Adhesion receptors were once grouped together with Class B receptors, members of the Adhesion family possess quite distinctive structural features. The amino (N)-termini of Adhesion receptors contain a range of structural domains including domains with adhesion properties and a GPCR Autoproteolysis-inducing (GAIN) domain adjacent to the first transmembrane region (Purcell & Hall, 2018). The Adhesion family can be subdivided into smaller receptor groups with distinct sequence characteristics, and

20 the current classification with nine subgroups is based on the nomenclature formed by NC-IUPHAR and the Adhesion GPCR Consortium (Hamann et al., 2015). The Class C/ Glutamate family includes receptors for gamma-aminobutyric acid (GABA), glutamate, calcium ions and sweet and umami taste (Lagerstrom & Schioth, 2008). These receptors contain an extracellular Venus fly trap ligand binding domain and function as obligatory dimers formed by two interacting family members (Jones et al., 1998; Pin & Bettler, 2016). The smallest GPCR subfamily is the frizzled family containing ten frizzled receptors and one receptor (Fredriksson et al., 2003). The Frizzled receptors bind secreted Wnt glycoproteins, whereas Smoothened functions together with another membrane protein called Patched to mediate Hedgehog signaling (Bhanot et al., 1996; Murone, Rosenthal, & de Sauvage, 1999).

2.1.2 Structure

The structural understanding of GPCRs is founded upon resolved receptor crystal structures along with sequence-based receptor analysis. However, the crystal structures only represent static snapshots of individual receptor conformations and typically require structural modifications that stabilize the receptor. Therefore, the understanding of GPCR dynamics requires crystal structures of several conformations combined with complementary spectroscopic and computational methods (Gusach et al., 2020; Latorraca, Venkatakrishnan, & Dror, 2017). The first high resolution crystal structure of a GPCR, that of rhodopsin, was published in 2000 (Palczewski et al., 2000). Thereafter, the number of GPCR structures have increased rapidly owing to major developments in protein engineering and crystallographic techniques. According to the GPCR database (http://gpcrdb.org), a key database that maintains information about GPCRs (Munk et al., 2016; Munk et al., 2019), more than 400 crystal structures of over 70 distinct receptors have been solved to date. Most of these structures are of Class A/ Rhodopsin receptors, although some examples from other families exist. The majority of GPCR structures represent inactive receptor conformations. Nonetheless, the most intensively studied receptors have yielded structures in active and intermediate as well as inactive conformations (Munk et al., 2019). Moreover, structures in complex with several ligand types and intracellular signaling components, such as G proteins and arrestins, have emerged (Hilger, Masureel, & Kobilka, 2018). The structure of GPCRs is characterized by seven membrane-spanning hydrophobic α-helical segments (Fig. 1A). These TM regions are connected by

21 three ECLs and three ICLs. The TM domains are organized to form a helical bundle (Fig 1B). This structure can be generally divided into three parts with distinct roles in the receptor activation and signaling. The extracellular region is formed by the N-terminus and ECLs, and in many GPCRs it modulates ligand access. The TM domains form the structural core that participates in ligand binding and undergoes conformational rearrangements that mediate the extracellular signal to the intracellular side. The intracellular region consists of ICLs and the carboxyl (C)- terminal tail that are responsible for interactions with intracellular signaling proteins (Venkatakrishnan et al., 2013).

Fig. 1. Schematic structure of GPCRs. A) GPCRs share a common structure of seven α- helical transmembrane (TM) domains that are connected by extracellular loops (ECL1- 3) and intracellular loops (ICL1-3). The amino (N)- and carboxyl (C)-terminal domains are located on the extracellular and intracellular sides, respectively. The C-terminal tail contains a short α-helical region that is typically followed by palmitoylated cysteines (zigzag line). The conserved microdomains in Class A receptors with key amino acid residues (DRY, CWxP and NPxxY) and the intramolecular disulfide bond between TM3 and ECL2 are also indicated (red). B) The TM helices form a barrel-like bundle. Certain residues, such as prolines, create kinks in the α-helices. C) Extracellular view of the TM domains. The conserved interhelical contacts in class A receptors are presented with red lines (Venkatakrishnan et al., 2013). The line thickness is proportional to the number of contacts between TM helices. Modified from Weis & Kobilka, 2018 and Venkatakrishnan et al., 2013.

22 Extracellular domains

The N-terminal domains are the most variable regions in GPCRs. Apart from Class A receptors, GPCRs generally possess long structured N-termini that play an important role in ligand recognition (Fredriksson et al., 2003; Venkatakrishnan et al., 2013). The peptide ligands of Class B GPCRs interact with the extracellular domains of the receptor, but also enter into the TM bundle (de Graaf et al., 2017). The Venus fly trap ligand binding site in Class C GPCRs is formed by two lobes that are separated by a cavity where the ligand binds (Geng, Bush, Mosyak, Wang, & Fan, 2013; Kunishima et al., 2000; Leach & Gregory, 2017). In the Frizzled family receptors, N-terminal cysteine-rich regions are involved in ligand binding (Byrne et al., 2016; Janda, Waghray, Levin, Thomas, & Garcia, 2012; Schulte, 2010). In the Adhesion receptors, in contrast, the N-terminal GAIN domain is a general feature that is only missing in adhesion GPCR A1 (Arac et al., 2012; Purcell & Hall, 2018). This domain mediates autoproteolytic cleavage of the receptor N- terminus and in some cases reveals an N-terminal tethered ligand sequence that can activate the receptor (Purcell & Hall, 2018; Vizurraga, Adhikari, Yeung, Yu, & Tall, 2020). Although most of the Class A receptors have relatively short N-terminal tails without common functional domains, some exceptions exist. Protease-activated receptors (PARs) contain specific proteolytic cleavage sites in their N-termini. As with Adhesion receptors, these receptors can be activated by a tethered peptide ligand exposed after the proteolytic cleavage of the receptor N-terminus (Hamilton & Trejo, 2017; Vu, Hung, Wheaton, & Coughlin, 1991). Leucine-rich repeat- containing GPCRs, such as glycoprotein hormone receptors, form another exceptional group of Class A GPCRs that contain large N-termini with conserved domains. These receptors encompass N-terminal leucine-rich repeats involved in ligand binding, and some receptors also carry a low-density lipoprotein class A module (Hsu et al., 2000; Petrie, Lagaida, Sethi, Bathgate, & Gooley, 2015). However, in most Class A GPCRs, ECLs and TM domains play more important roles in ligand interactions (Lagerstrom & Schioth, 2008; Venkatakrishnan et al., 2013). The ECLs can either leave the ligand binding pocket in the TM region water- accessible or form a lid-like structure (Venkatakrishnan et al., 2013). The ECL2 is relatively large and may contain secondary structural elements such as α-helices and β-sheets. In contrast, ECL1 and ECL3 are shorter and usually lack secondary structure (Unal & Karnik, 2012; Venkatakrishnan et al., 2013).

23 The extracellular domains of GPCRs are often modified by co- and post- translational modifications occurring during receptor synthesis in the endoplasmic reticulum (ER) and transport in the early secretory pathway. Disulfide bonds between cysteine residues are typical modifications in GPCRs that stabilize the structure. Most GPCRs contain a highly conserved disulfide bridge between ECL2 and TM3 (Fig 1A) (Venkatakrishnan et al., 2013). Additionally, certain subfamily- specific disulfide bonds exist, especially in GPCRs with long N-termini (Lagerstrom & Schioth, 2008; Unal & Karnik, 2012). Glycosylation is another common modification in the extracellular domains of GPCRs and typically contributes to receptor folding and trafficking to the cell surface (Goth, Petaja-Repo, & Rosenkilde, 2020). The N-terminal tail is also important for cell surface expression of certain GPCRs such as the dopamine D2 receptor (D2R), endothelin B receptor (ETBR) and α2B- (Cho et al., 2012; C. Dong & Wu, 2006; Grantcharova et al., 2002). On the other hand, truncation of the N-terminal tail reportedly increases cell surface expression of GPR37 and 1 (Andersson, D'Antona, Kendall, Von Heijne, & Chin, 2003; Dunham, Meyer, Garcia, & Hall, 2009) suggesting an opposite role of this domain. The N-terminal domain can also impact receptor activity and downstream signaling as demonstrated for the 5-hydroxytryptamine receptor 2B and melanocortin-4 receptor (Belmer et al., 2014; Ersoy et al., 2012).

Transmembrane domains

The TM domains represent the most conserved region in the GPCR structure. These domains form the structural core of GPCRs (Fig. 1C) that is supported by a conserved network of noncovalent contacts between the helices. Especially TM3, which forms consensus contacts with all TM domains except TM1 and TM7 in Class A GPCRs, appears to play an important role maintaining GPCR structure. The TM region is also important for ligand binding in Class A and B receptors. In Class A GPCRs, residues in TMs 3, 6 and 7 provide important contacts for ligand binding. Residues in other TMs may also participate in ligand interactions to varying extents. Nevertheless, the amino acid residues at the topologically equivalent positions in the ligand binding pocket vary between different receptors, thus contributing to the ligand selectivity of individual receptors (Katritch, Cherezov, & Stevens, 2012; Venkatakrishnan et al., 2013). The class A GPCRs contain certain conserved structural motifs in their TM regions that undergo changes in residue orientation and contacts upon receptor

24 activation. The Asp/Glu-Arg-Tyr (D/ERY) motif is located at the boundary of TM3 and ICL2 (Fig. 1A) and is involved in G protein binding as well as the conformational changes related to receptor activation. Especially the highly conserved arginine residue plays an important role in this motif. It forms a salt bridge with the neighboring aspartic-/glutamic acid, and in some GPCRs also interacts with TM6 under inactive conditions (Katritch, Cherezov, & Stevens, 2013). The Cys-Trp-x-Pro (CWxP, where x denotes any amino acid) sequence is found in TM6 (Fig. 1A) and is located at the bottom of the main ligand binding pocket. The conserved tryptophan in this motif functions as a ligand-induced conformational switch (Nygaard, Frimurer, Holst, Rosenkilde, & Schwartz, 2009). The Asn-Pro-x-x-Tyr-x-Phe (NPxxY) motif is located at the cytoplasmic end of TM7 (Fig. 1A). The conserved tyrosine residue of this motif forms distinct interactions with other TM helices in inactive and active conformations and contributes to G protein activation (Angel, Chance, & Palczewski, 2009; Katritch et al., 2013). Additionally, an allosteric sodium binding site in the middle of the TM bundle and a connector motif formed between conserved proline, isoleucine and phenylalanine (PIF) residues in TM5, TM3 and TM6, respectively, represent important structural features involved in Class A GPCR activation (Katritch et al., 2014; Schonegge et al., 2017; Zhou et al., 2019). Although the main emphasis of GPCR structural studies has been on Class A receptors, certain subfamily-specific conserved and functionally important residues are also found in other GPCR families (Binet et al., 2007; de Graaf et al., 2017; Pin & Bettler, 2016; Schulte & Kozielewicz, 2020). GPCR activation involves reorganization of the residue contacts in the TM helices and conformational changes in the receptor (Venkatakrishnan et al. 2013). There are two alternative models for GPCR activation, both of which are compatible with existing information (Deupi & Kobilka, 2010; Kenakin, 1995). In the conformational selection scenario, the receptor alternates between various active and inactive conformations and the ligand binds to and stabilizes the active receptor conformations. In the induced-fit mechanism, in contrast, conformational changes in the receptor are induced by ligand binding (Deupi & Kobilka, 2010; Hunyady, Vauquelin, & Vanderheyden, 2003; Kenakin, 1995). In Class A GPCRs, the reported structural changes upon receptor activation include a rotation of TM5, an inward movement of TM7 toward TM3 and an outward movement of the cytoplasmic end of TM6. The movement of TM6 opens the cytoplasmic side of the receptor revealing important residues for G protein coupling (Venkatakrishnan et al., 2016; Zhou et al., 2019). Some general features of receptor activation, such as

25 the outward movement of TM6, are believed to apply to different GPCR subfamilies. However, the precise mechanisms vary between receptor families and individual receptors within the same subfamily (Hilger et al., 2020; Venkatakrishnan et al., 2016).

Intracellular domains

The intracellular domains of GPCRs show remarkable diversity both between and within different receptor families. In Class A GPCRs, the cytoplasmic tail and ICL3 are typically long, and are highly disordered regions in the receptors (Jaakola, Prilusky, Sussman, & Goldman, 2005; Venkatakrishnan et al., 2014). However, many receptors contain a short amphipathic helix called helix 8 in the cytoplasmic domain close to TM7 (Venkatakrishnan et al., 2013). The cysteines at the C- terminal end of this helix are usually modified by palmitoylation, which functions as a membrane anchor (Qanbar & Bouvier, 2003). The ICL1 and ICL2 are shorter and may contain α-helical turns or unstructured stretches (Venkatakrishnan et al., 2013). The C-terminal tails and ICLs of GPCRs serve as sites for interactions with diverse regulatory proteins. For example, receptors may contain motifs that mediate sorting in GPCR trafficking pathways or control receptor anchoring to separate regions of the plasma membrane. Various interacting proteins can also affect the signaling mechanism of the receptor (Marchese, Paing, Temple, & Trejo, 2008; Ritter & Hall, 2009). In many cases, receptor interactions are mediated by postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-binding motifs that are present in several GPCRs (Dunn & Ferguson, 2015). Additionally, the intracellular regions of GPCRs can be subjected to various post- translational modifications. Phosphorylation of serine and threonine residues in the intracellular domains is a common mechanism to regulate the GPCR signaling response and receptor internalization from the plasma membrane (Rajagopal & Shenoy, 2018). The intracellular domains can also be modulated by other types of post-translational modifications such as ubiquitination, nitrosylation or hydroxylation (Daaka, 2012; Dores & Trejo, 2019; Xie et al., 2009).

2.1.3 Biosynthesis and trafficking in cells

Although the plasma membrane is considered the primary location of functionally active GPCRs, these receptors begin their lifecycles in the ER (Marino, Walser,

26 Poms, & Zerbe, 2018; Skach, 2009). Following stringent quality control in the ER, properly folded receptors traffic to the cell surface. The number of signaling receptors available at the plasma membrane is regulated by ER export and delivery to the cell surface along with internalization and degradation (Duvernay, Filipeanu, & Wu, 2005). Membrane trafficking is also intimately involved in the subcellular organization of receptor signaling, and some GPCRs can maintain functional activity following internalization (Eichel & von Zastrow, 2018).

Biosynthesis, folding and ER quality control

The synthesis and folding of multi-spanning membrane proteins, such as GPCRs, are complex processes that are not fully understood. The synthesis of GPCRs occurs in the ribosomes of the ER membrane (Fig. 2A). GPCRs are cotranslationally translocated into the ER through a protein-conducting translocon channel starting from the N-terminus of the nascent polypeptide chain. The α- helical TM segments fold independently and are inserted into the ER membrane (Ellgaard, McCaul, Chatsisvili, & Braakman, 2016; Marino et al., 2018; Skach, 2009). Some GPCRs carry N-terminal signal peptides that mediate receptor targeting and integration into the ER and are cleaved off following ER insertion (Schulein, Westendorf, Krause, & Rosenthal, 2012). Signal peptides are important for certain receptors that carry rapidly and stably folded domains in their N-termini or have large N-terminal tails (Kochl et al., 2002; Schulein et al., 2012). However, in most GPCRs, ER targeting is achieved by signal-anchor sequences in the TM domains (Schulein et al., 2012; Wallin & Vonheijne, 1995). Protein folding begins already during receptor translocation and is assisted by co-translational modifications, such as disulfide bond formation and N-glycosylation, and various molecular chaperones present in the ER (C. M. Dong, Filipeanu, Duvernay, & Wu, 2007; Tao & Conn, 2014). Protein folding is a slow process that is prone to errors, and some proteins fail to achieve their native fold. The ER folding and quality control machinery ensures that proteins are correctly folded before they exit the ER. Molecular chaperones and folding factors present in the ER recognize misfolded proteins and help them to achieve their native conformations. Lectin chaperones calnexin and calreticulin interact with N-glycosylated proteins, including GPCRs, to assist their folding (Lamriben, Graham, Adams, & Hebert, 2016; Ruddock & Molinari, 2006; Tao & Conn, 2014). GPCRs are also known to interact with the binding immunoglobulin protein (BiP) and certain other chaperones of the heat shock protein family (Tao &

27 Conn, 2014). Additionally, folding enzymes, such as protein disulfide isomerases and peptidyl-prolyl isomerases, participate in the ER quality control of GPCRs by catalyzing co-and post-translational modifications that are important for protein folding (Ellgaard et al., 2016; Tao & Conn, 2014). Intriguingly, certain native GPCRs are known to fold inefficiently despite the chaperones and folding factors present in the ER. Therefore, a substantial fraction of these receptors is retained in the ER and never traffics to the plasma membrane (Petäjä-Repo, Hogue, Laperrière, Walker, & Bouvier, 2000; Pietila et al., 2005). Furthermore, ER retention and aggregation of misfolded GPCRs have been associated with some pathological conditions. For example, misfolding and ER retention of mutated V2 vasopressin receptors cause nephrogenic diabetes insipidus (Birnbaumer, Gilbert, & Rosenthal,

1994) , whereas retention causing mutations in the ETBR lead to Hirschsprung’s disease (Fuchs et al., 2001). Similarly, mutations causing misfolding of melanocortin-4 receptor are associated with severe early-onset obesity (Rene et al., 2010). Moreover, ER retention of the frizzled-4 receptor and rhodopsin are implicated in familial exudative vitreoretinopathy and retinitis pigmentosa, respectively (Noorwez et al., 2004; Robitaille et al., 2002). To avoid aggregation of misfolded proteins and consequent cellular stress, receptors that fail to achieve their native configuration are targeted for degradation in a series of pathways collectively called the ER-associated degradation (ERAD) (Fig. 2B). The ERAD substrates are recognized in the ER and directed away from the folding machinery. These proteins are then retrotranslocated across the ER membrane and dislocated to the cytosol where they are degraded by a protease complex termed the 26S proteasome (Christianson & Ye, 2014; Wu & Rapoport, 2018). Covalent attachment of ubiquitin to lysine residues in the C-terminal tail and ICLs of GPCRs serves as a sorting signal that ultimately directs receptors for proteasomal degradation (Dores & Trejo, 2019). The ubiquitination mechanism involves the sequential action of ubiquitin-activating enzyme (E1), ubiquitin- conjugating enzyme (E2) and ubiquitin ligase (E3) on the cytoplasmic side of the ER membrane (Christianson & Ye, 2014; Wu & Rapoport, 2018). In some cases, ERAD-resistant misfolded proteins may be directed to alternative degradative pathways in the endo-lysosomal compartments (Fregno & Molinari, 2019; Houck et al., 2014). The accumulation of unfolded or misfolded proteins can also turn on the unfolded protein response, a network of adaptive mechanisms to cope with ER stress (Hetz, 2012). These signaling pathways are often involved in neurogenerative diseases characterized by protein aggregation and ER stress (Hetz & Saxena, 2017).

28 Fig. 2. Biosynthesis and trafficking of GPCRs. GPCRs are synthesized and begin to fold cotranslationally in the endoplasmic reticulum (ER) (A). If the receptors are not able to pass the ER quality control and fold properly, they are targeted to the proteasome for degradation (B). The correctly folded and assembled receptors traffic through the ER- Golgi intermediate compartment (ERGIC), Golgi and trans-Golgi network (TGN) (C) to the plasma membrane that is the primary location of GPCR signaling (D). Receptor phosphorylation by protein kinases and interaction with arrestins promote internalization via clathrin-coated pits (E). Clathrin-coated vesicles detach from the plasma membrane and, following uncoating, fuse with the sorting endosomes (F). After internalization, GPCRs can recycle back to the plasma membrane either directly or via retrograde transport to the TGN (G). The receptors may also continue to initiate signaling cascades from endosomes or the TGN following internalization. Alternatively, GPCRs may be sorted into the intraluminal vesicles of late endosomes/multivesicular bodies (MVBs) that fuse with lysosomes to mediate receptor degradation (H). Modified from Hanyaloglu & von Zastrow, 2008 and Tao & Conn, 2014.

29 Trafficking to the cell surface

Properly folded receptors are able to exit the ER and traffic to the cell surface. GPCRs are transported through the ER-Golgi intermediate compartment (ERGIC) to the Golgi in ER-derived coatomer protein II-coated vesicles (Fig. 2C). In the Golgi, receptors traffic from the cis-Golgi, which faces the ER, through the medial Golgi to the trans-Golgi network (TGN). Finally, the receptors are transported from the TGN to the plasma membrane (Fig. 2D) (C. M. Dong et al., 2007). Trafficking in the ERGIC and Golgi can occur in the anterograde direction toward the plasma membrane, but also in the retrograde direction back toward the ER (Young, Wertman, & Dupre, 2015). The ER exit and forward trafficking of GPCRs to the plasma membrane are highly coordinated processes. These mechanisms are regulated by post-translational modifications, interacting proteins, specific motifs in the receptor sequence, and in some cases receptor dimerization (Young et al., 2015; M. X. Zhang & Wu, 2019). The regulatory proteins involved in the ER export and trafficking of GPCRs include chaperones, gatekeepers, escort proteins, proteins of the transport machinery and signaling proteins (M. X. Zhang & Wu, 2019). Rab proteins comprise a family of guanine triphosphatases that are involved in membrane trafficking. GPCR anterograde and retrograde trafficking can be regulated by Rabs 1, 2, 6 and 8 that are generally implicated in the vesicular transport between ER, ERGIC and Golgi (Young et al., 2015; M. X. Zhang & Wu, 2019). Additionally, recent evidence suggests that Rab43 might be a GPCR-specific regulator of the ER- Golgi transport (C. M. Li et al., 2017). There are also several receptor-specific proteins involved in GPCR cell surface trafficking, such as receptor activity- modifying proteins and homer proteins, along with many other types of receptor- interacting proteins (C. M. Dong et al., 2007; M. X. Zhang & Wu, 2019). Furthermore, GPCR ER export can be facilitated by pharmacological chaperones. These small membrane-permeable molecules bind specifically to their target proteins and promote correct folding and trafficking (Morello, Petaja-Repo, Bichet, & Bouvier, 2000). Pharmacological chaperones also have therapeutic relevance since many diseases involve ER retention of misfolded proteins (Conn et al., 2014). For instance, pharmacological chaperones have been used to rescue folding and cell-surface delivery of mutant forms of V2 that cause nephrogenic diabetes insipidus (Morello, Salahpour, et al., 2000). Similarly, the folding and trafficking of the mutated melanocortin-4 receptor that is implicated in obesity and rhodopsin and frizzled-4 mutants associated with retinal diseases were

30 improved by pharmacological chaperones (Fan & Tao, 2009; Generoso et al., 2015; Noorwez et al., 2003). Moreover, a similar strategy has been used to rescue the cell surface delivery of mutants of the luteinizing hormone receptor implicated in Leydig cell hypoplasia and the gonadotropin-releasing hormone receptor involved in hypogonadism (Janovick et al., 2009; Newton et al., 2011).

Glycosylation

As mentioned earlier, GPCRs are often subjected to co- and posttranslational modifications during their synthesis and trafficking in the secretory pathway. N- linked glycosylation is a common modification of GPCRs, and most receptors carry at least one N-glycosylation site in their extracellular domains. N-glycosylation is involved in protein folding and quality control, and in many GPCRs plays a role in receptor trafficking to the cell surface (Goth et al., 2020). The core structure of N- glycans is composed of two N-acetylglucosamines, nine mannoses and three glucoses. This core glycan is co-translationally transferred from a lipid donor to the asparagine residue of the Asn-x-Ser/Thr (where x is any amino acid except proline) consensus motif in the target polypeptide chain by the oligosaccharyltransferase enzyme. The N-glycans are then subjected to trimming by glycosidases that remove glucose and mannose residues from the core structure. This glycan trimming is intimately related to protein quality control and ERAD to ensure that only properly folded glycoproteins exit the ER (Aebi, Bernasconi, Clerc, & Molinari, 2010; Helenius & Aebi, 2004). The N-glycans can be divided into three general classes. The N-glycans in the ER localized glycoproteins are high-mannose type glycans containing only mannose residues linked to the core N-acetylglucosamines. After the glycoprotein leaves the ER, the glycans are further modified by glycosidases and glycosyltransferases in the Golgi to form mature hybrid or complex type N- glycans containing also other types of sugar moieties (Reily, Stewart, Renfrow, & Novak, 2019). O-glycosylation is another type of glycan modification present in some GPCRs (Goth et al., 2020; Goth, Vakhrushev, Joshi, Clausen, & Schjoldager, 2018). In contrast to N-glycosylation, O-glycosylation occurs in the Golgi after protein folding. Membrane proteins, including GPCRs, are typically modified by N- acetylgalactosamine O-glycosylation also known as mucin-type glycosylation. This modification occurs on a serine or threonine, or in rare cases tyrosine, residue of the target protein (Goth et al., 2020; Reily et al., 2019). Although a number of GPCRs have been demonstrated to carry O-glycans, the functional consequences

31 of O-glycosylation are poorly understood (Goth et al., 2020). The role of O- glycosylation has been implicated in the ligand binding affinity of prostaglandin receptors and turnover of the δ- (Lackman et al., 2018; Morii & Watanabe, 1992), but for most GPCRs, the functional role of this modification remains unexplored. Interestingly, O-glycosylation appears to regulate proteolytic cleavage of certain membrane proteins and might have a similar role in GPCRs that are subjected to proteolytic cleavage in their N-terminal domains (Goth et al., 2018). This kind of mechanism has been recently demonstrated for the β1-adrenergic receptor (Goth et al., 2017; Park, Reddy, Wallukat, Xiang, & Steinberg, 2017), and may be more generally involved in the regulation of proteolytically processed GPCRs.

Endocytic trafficking

The activity of GPCRs can be temporally and spatially regulated by internalization from the cell surface via endocytosis. This mechanism is also associated with attenuation of receptor-mediated responses at the plasma membrane (described in more detail in chapter 2.1.4) (Hanyaloglu & von Zastrow, 2008). Receptor internalization occurs in membrane-derived vesicles that are usually coated with clathrin (Fig. 2E). GPCRs are sorted into clathrin-coated pits by interaction with adaptor proteins that encompass cargo- and clathrin-binding domains. The cargo- containing endosomes are then formed by dynamin-dependent vesicle scission (Weinberg & Puthenveedu, 2019). After internalization, GPCRs are subjected to endosomal sorting that dictates their destination (Fig. 2F). Receptors can be recycled back to the cell surface (Fig. 2G) or targeted for lysosomal proteolytic degradation (Fig. 2H) (Hanyaloglu & von Zastrow, 2008; Pavlos & Friedman, 2017). GPCRs may also continue to signal from the intracellular compartments after internalization (Lobingier & von Zastrow, 2019). Internalization and endosomal trafficking involve adaptor proteins, such as the adaptor protein2 complex and GPCR-associated sorting proteins, that interact with specific motifs in the receptor C-terminal tail (Magalhaes, Dunn, & Ferguson, 2012; Marchese et al., 2008) Endosomal trafficking of GPCRs also involves various guanine triphosphatases (Magalhaes et al., 2012). These include Rab family proteins Rab5 for early endosomes, Rab7 for late endosomes and Rab4 and 11 for recycling endosomes (Pavlos & Friedman, 2017). Targeting for lysosomal degradation is typically mediated by ubiquitination and interactions with the endosomal sorting

32 complex required for transport (ESCRT) machinery, but ubiquitin can also regulate GPCR sorting beyond degradation (Dores & Trejo, 2019).

2.1.4 Signaling

GPCRs are classically perceived as cell surface receptors that mediate signaling from the extracellular space to the intracellular environment. As the name of the receptor family implies, the intracellular signaling response is usually triggered by activation of heterotrimeric G proteins. However, research during the past years has revealed that GPCR signaling is a far more multidimensional process than originally anticipated. Thus, it is now known that GPCRs can signal in a manner that is neither restricted to plasma membrane localization nor confined to signal transduction via heterotrimeric G proteins. It is also evident that GPCRs do not function as simple on/off switches that alternate between active and inactive conformations. Rather, they are extremely dynamic proteins that can sample various active and inactive conformational states. Interactions with ligands and intracellular signaling proteins, however, alter the conformational dynamics of GPCRs and can stabilize distinct receptor conformations (Fig. 3A) (Latorraca et al., 2017; Wingler & Lefkowitz, 2020).

Ligands and their binding sites

GPCR ligands are typically classified according to the effect they exert on the conformational landscape and functional properties of the receptor (Fig. 3B). The extent to which a ligand can activate a particular signaling pathway is described by the term ligand efficacy (Weis & Kobilka, 2018). Most GPCRs show a significant degree of basal ligand-independent activity arising from the equilibrium between active and inactive receptor conformations (Katritch et al., 2013). Full and partial agonists are ligands that stimulate receptor activity above the basal level by stabilizing active receptor conformations. However, partial agonists induce weaker maximal activity than full agonists even at saturating concentrations. Full or partial inverse agonists, in contrast, stabilize inactive receptor conformations and reduce receptor activity below the basal level. Neutral antagonists do not alter the equilibrium between active and inactive receptor states, and thus do not produce a response. Instead, they can bind to the same ligand-binding site with other ligands and antagonize their activities (Weis & Kobilka, 2018). The consequences of receptor-ligand binding are further complicated by the concept of biased agonism

33 (Fig. 3C). GPCRs are typically able to activate multiple downstream signaling pathways. Biased ligands, however, show selectivity toward certain pathway(s) over the others (J. S. Smith, Lefkowitz, & Rajagopal, 2018; Weis & Kobilka, 2018).

Fig. 3. The effect of ligands on GPCR signaling. Conformational dynamics of GPCRs can be illustrated by energy landscapes that are based on the information obtained by structural biological methods (Costa-Neto, Parreiras, & Bouvier, 2016; Deupi & Kobilka, 2010). A) As this hypothetical landscape shows, GPCRs can explore various inactive- like (R, R’, R’’) and active-like (R*, R**) conformational states. In the absence of a ligand, the energy barrier to achieve active states is high making their occurrence unlikely. Ligand binding decreases the energy required for active states, and binding of the intracellular G protein to the receptor-ligand complex further alters the energy landscape and stabilizes the fully active state. B) Efficacy of GPCR ligands. Distinct types of ligands differ in their ability to alter GPCR activity. C) Biased ligands preferentially stimulate certain signaling pathway over the other(s). Downstream signaling can be mediated by distinct subtypes of G proteins or other types of transducers such as arrestins. Modified from Deupi & Kobilka, 2010, Weis & Kobilka, 2018 and Wootten et al., 2018.

34 Endogenous ligands interact with their cognate receptors at the primary binding site called the orthosteric site. Additionally, GPCRs can be subject to regulation by allosteric modulators that bind to distinct receptor sites termed allosteric sites (Thal, Glukhova, Sexton, & Christopoulos, 2018; Wootten, Christopoulos, & Sexton, 2013). Diverse factors, ranging from protein partners to ions and lipids, can act as allosteric modulators (Gusach et al., 2020; Wootten et al., 2018). They can alter the binding and/or function of the ligands at the orthosteric site, and they are generally classified as positive or negative modulators based on their effect on the orthosteric ligand action. However, some molecules act as neutral or silent allosteric modulators that do not have any net effect on the binding or efficacy of the orthosteric ligand. Nonetheless, they can prevent the binding of other modulators to the same allosteric site (Thal et al., 2018; Wootten et al., 2013). In certain cases, orthosteric and allosteric binding sites are located in close proximity, and they can be simultaneously targeted by a single ligand termed a bitopic ligand (Valant, Robert Lane, Sexton, & Christopoulos, 2012).

Downstream signaling effectors

GPCRs initiate intracellular signaling responses by interacting with various transducers at the cytoplasmic site. As their name implies, GPCRs typically signal via heterotrimeric G proteins, which consist of three subunits: Gα, Gβ and Gγ (Syrovatkina, Alegre, Dey, & Huang, 2016). The Gα subunit is responsible for the binding of guanine nucleotides, i.e., guanosine diphosphate (GDP) and guanosine triphosphate (GTP). In an inactive GDP-bound form, Gα forms a heterotrimer with the other two G protein subunits, whereas the active GTP-bound conformation dissociates from the Gβ and Gγ subunits. While Gα subunits can function independently, Gβ and Gγ subunits form heterodimers that signal as a single functional unit (Oldham & Hamm, 2008; Schmidt, Thomas, Levine, & Neer, 1992; Syrovatkina et al., 2016). G proteins are usually classified based on the sequence and functional similarities of their α subunits. The Gα proteins can be grouped into four distinct families: Gαs, Gαi/o, Gαq/11 and Gα12/13 (Table 2). Members of these families trigger various signaling pathways by engaging with diverse downstream effectors, which in turn regulate the generation of second messengers (reviewed in Syrovatkina et al., 2016; Wootten et al., 2018). Table 2 summarizes some of the well-defined Gα effectors. While Gβγ dimers were originally thought to function mainly as negative regulators of Gα subunits, more recent evidence shows that they can directly

35 modulate several effectors (Table 2). In contrast to Gα proteins, which engage with relatively specific sets of effector proteins, Gβγ subunits have been reported to interact with a wide range of structurally unrelated targets without clearly specified binding determinants (reviewed in Dupre, Robitaille, Rebois, & Hebert, 2009; Smrcka & Fisher, 2019).

Table 2. Classification, main effectors and downstream effects of G protein subunits.

Family Subtypes Effectors Downstream effects

Gαs αs, αsXL, αolf Adenylyl cyclase↑ cAMP↑, protein kinase A↑ Src tyrosine kinase Tyrosine phosphorylation

Gαi/o αi1-3, αt1-2, αo1-2, αgust, αz Adenylyl cyclase↓ cAMP↓, protein kinase A↓ Src tyrosine kinase Tyrosine phosphorylation cGMP phosphodiesterase↑ cGMP hydrolysis 2+ Gαq/11 αq, α11, α14, α15/16 PhospholipaseC-β IP3↑, DAG↑, [Ca ]i↑, protein kinase C↑ p63RhoGEF↑ RhoA GTPase↑

Gα12/13 α12, α13 RhoGEFs↑ RhoA GTPase↑

Gβ/ Gγ* β1-5/ γ1-12 Adenylyl cyclase↑ cAMP↑, protein kinase A↑ GPCR kinases GPCR phosphorylation 2+ PhospholipaseC-β↑ IP3↑, DAG↑, [Ca ]i↑, protein kinase C↑ Ca2+ and K+ channels Cellular ion concentration PI3 kinases PI phosphorylation * Gβγ subunits signal as functional dimers. ↑, stimulation/ increase; ↓ inhibition/ decrease. Abbreviations: 2+ cAMP, cyclic adenosinemonophosphate; [Ca ]I, intracellular calcium concentration; cGMP, cyclic guanosine monophosphate; DAG, diacylglycerol; GTPase, guanosine triphosphate hydrolase; IP3, inositol triphosphate; PI, phosphatidylinositol; RhoGEF, Rho-guanine nucleotide exchange factor (Milligan & Kostenis, 2006; Syrovatkina et al., 2016; Wettschureck & Offermanns, 2005)

In addition to heterotrimeric G proteins, GPCR signaling can be mediated by arrestins, proteins that were originally named for their ability to “arrest” G protein- mediated signaling (DeWire, Ahn, Lefkowitz, & Shenoy, 2007). Mammals express four arrestins: arrestin-1/ visual arrestin, arrestin-2/ β-arrestin-1, arrestin-3/ β- arrestin-2 and arrestin-4/ cone arrestin. While the expression pattern of arrestin-1 and arrestin-4 is restricted mainly to cells of the visual system, the two β-arrestins are ubiquitously expressed and therefore able to engage with most GPCRs (Gurevich & Gurevich, 2019). Arrestins have been demonstrated to regulate various signaling proteins including those involved in distinct cascades of mitogen activated protein kinases: extracellular signaling kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases and p38 kinases (reviewed in DeWire et al., 2007; Jean-Charles, Kaur, & Shenoy, 2017). GPCR signaling via arrestins is often called G protein-

36 independent signaling. However, current evidence indicates that arrestin-mediated GPCR signaling may require initial G protein activation (Grundmann et al., 2018; Wootten et al., 2018), and thus, it remains unclear to what extent arrestins can function with GPCRs in a truly G protein-independent manner.

The canonical G protein-mediated signaling pathway

The canonical mechanism of GPCR signaling relies on signal transduction via heterotrimeric G proteins (Fig. 4A). Receptor activation promotes engagement with the GDP-bound G protein heterotrimer (Hilger et al., 2018; Oldham & Hamm, 2008). Subsequent GDP dissociation from the Gα subunit is the rate-limiting step in the G protein activation cycle and is rapidly followed by GTP binding (Ferguson, Higashijima, Smigel, & Gilman, 1986; Hilger et al., 2018). Consequently, conformational changes in the Gα subunit trigger dissociation of Gα from the Gβγ dimer. Both Gα and Gβγ interact with various downstream effectors to induce intracellular signaling cascades. The G protein activation cycle is finally terminated by GTP hydrolysis to GDP in the Gα subunit prompting reassociation of the G protein heterotrimer (Hilger et al., 2018; Johnston & Siderovski, 2007).

37 Fig. 4. Mechanisms of GPCR signaling. A) the canonical GPCR signaling pathway is mediated by heterotrimeric G proteins. G protein binding to the activated receptor leads to dissociation of G protein subunits and activation of downstream effectors such as adenylyl cyclase (AC) and ion channels. B) Biased signaling involves selective activation of a certain downstream signaling pathway that is preferred over the other pathways. C) GPCR signaling and localization can be altered by dimerization or formation of higher order oligomers. These oligomers can be classified into receptor homomers consisting of identical receptors or heteromers involving different GPCR subtypes. D) Adhesion family receptors and PARs can be activated by tethered peptide agonists that are revealed after proteolytic cleavage of the receptor N-terminal domain. E) GPCRs may also signal from intracellular compartments, such as endosomes and the Golgi complex, following receptor internalization or, in some cases, prior to trafficking to the cell surface. Modified from Hamilton & Trejo, 2017, Soave, Briddon, Hill, & Stoddart, 2020, Weis & Kobilka, 2018 and Wootten et al., 2018.

38 GPCR signaling beyond the canonical mechanism

GPCR research during the past couple of decades has started to reveal the multifaceted nature of their signaling mechanisms. Thus, the concept of biased signaling or functional selectivity has been established to exemplify the complexity of GPCR signaling outcomes (Urban et al., 2007). As mentioned earlier, biased agonists can stabilize receptor conformations that selectively favor certain signaling pathways over the others (Fig. 4B) (J. S. Smith et al., 2018; Weis & Kobilka, 2018). Additionally, several other factors can alter the receptor conformational landscape, and thus contribute to biased signaling. For example, membrane composition, receptor assembly into membrane microdomains, availability of different effectors and interacting proteins, allosteric modulators, kinetics of ligand-receptor interaction and genetic variation in receptor structure and expression can all affect the signaling profiles of GPCRs (Costa-Neto et al., 2016; J. S. Smith et al., 2018; Wootten et al., 2018). Furthermore, receptor function can be modified by assembly into multiprotein complexes with GPCRs or other types of proteins. Indeed, several GPCRs are reported to form dimers or higher order oligomers (Fig. 4C). Oligomerization can alter receptor trafficking and function in various ways, and in some cases, formation of receptor dimers is obligatory for G protein activation and biological function of the GPCR (Bouvier, 2001; Milligan, 2001; Pin & Bettler, 2016). Some GPCRs can also be activated by tethered agonists that are encrypted in the receptor N-terminal domain (Fig. 4D) (Hamilton & Trejo, 2017; Vizurraga et al., 2020). GPCR signaling from intracellular compartments is yet another concept that has gained a lot of interest among researchers recently (Fig. 4E). Endosomal GPCR signaling, in particular, has been a subject of intensive research, and the mechanism can involve both G protein and arrestin-mediated signaling pathways (Eichel & von Zastrow, 2018; Irannejad & von Zastrow, 2014; Sposini & Hanyaloglu, 2017). The G protein-mediated signaling results either from the prolonged activation of internalized plasma membrane receptors or from the recovered ability of receptors to activate G proteins in the endosomes (Irannejad & von Zastrow, 2014). Additionally, GPCR signaling activity has been detected in several other intracellular compartments including the Golgi and Golgi-associated membranes, ER, and even rather unexpected places such as mitochondria and nuclear membrane (Jong, Harmon, & O'Malley, 2018; Lobingier & von Zastrow, 2019; Plouffe, Thomsen, & Irannejad, 2020; Ribeiro-Oliveira et al., 2019). However, it is obvious that more research is still needed to determine to what extent these mechanisms

39 occur under physiological conditions as most results are obtained using cultured cell models (Lobingier & von Zastrow, 2019).

Termination of GPCR-mediated signaling

The GPCR activity is regulated by mechanisms of desensitization and downregulation that decrease receptor response to prolonged or repeated stimulation. GPCR desensitization involves kinases that phosphorylate serine and threonine residues in the intracellular domains of the receptor and arrestins that are recruited to the phosphorylated receptor to facilitate the desensitization process (Bouvier et al., 1988; Rajagopal & Shenoy, 2018). Receptor desensitization can occur in a homologous or heterologous manner and these mechanisms involve different types of kinases. While homologous desensitization takes place in response to agonists that act at a specific GPCR subtype, heterologous desensitization is a more general process involving simultaneous desensitization of several GPCR subtypes even in the absence of their cognate ligands (Kelly, Bailey, & Henderson, 2008). Homologous desensitization is mediated by GPCR kinases that specifically phosphorylate the activated receptors to promote arrestin recruitment. In contrast, heterologous desensitization is regulated by second messenger-dependent kinases, such as protein kinase A and protein kinase C, and can occur in an arrestin-independent manner (Kelly et al., 2008; Lefkowitz, 1998; Rajagopal & Shenoy, 2018). Phosphorylation of GPCRs and consequent β-arrestin recruitment facilitate receptor uncoupling from G proteins and promote interactions with the endocytic machinery. Internalized receptors are either dephosphorylated and recycled back to the cell surface for another round of stimulation or downregulated by lysosomal degradation (Ritter & Hall, 2009; Shenoy & Lefkowitz, 2011). The recently discovered noncanonical signaling pathways require also unconventional means of signal termination. Although the current knowledge of these mechanisms is incomplete, some evidence for distinct desensitization pathways has started to emerge (Eichel & von Zastrow, 2018). For instance, endosomal acidification, engagement with arrestin-like proteins and sequential action of certain protein trafficking machineries, such as the retromer complex, have been implicated in the termination of endosomal GPCR signaling (Eichel & von Zastrow, 2018; Lobingier & von Zastrow, 2019; Pavlos & Friedman, 2017). Biased signaling can also alter GPCR engagement with proteins involved in receptor desensitization. For example, biased ligands can facilitate recruitment of

40 different GRK family members to mediate receptor phosphorylation, which in turn alters interactions with arrestins (Rajagopal & Shenoy, 2018).

2.1.5 Orphan GPCRs

Orphan GPCRs are receptors without an identified endogenous ligand. There are still approximately 100 GPCRs that are considered orphans (Table 1 shows the subdivision of orphan GPCRs by receptor families), and consequently, their functional mechanisms are not clearly defined (Alexander et al., 2019; Laschet et al., 2018). However, owing to the prominent role of GPCRs as drug targets, these receptors represent a great potential for novel therapeutic interventions (Civelli et al., 2013; Laschet et al., 2018). Therefore, ligand identification plays an important role in orphan GPCR research. To provide some clarity to the endeavors of receptor-ligand coupling, IUPHAR has created general guidelines to define when a GPCR can be considered deorphanized (Davenport et al., 2013). The primary criterion is reproducibility. Therefore, the activity of the proposed ligand at the receptor must be demonstrated by two or more peer-reviewed papers from independent research groups using potencies (i.e. concentrations required to produce an effect) compatible with a physiological function. Additional criteria include the use of diverse methods to test the ligand activity and sufficient availability of the potential endogenous ligand in tissues expressing its cognate receptor. Furthermore, receptor deletion and overexpression in animal models should cause a phenotype that is consistent with the proposed pharmacological effect of the ligand on the receptor (Davenport et al., 2013). From a historical viewpoint, ligands of GPCRs have been discovered before their cognate receptors (reviewed in Civelli et al., 2013; Laschet et al., 2018). However, the rapid development of molecular biological methods in the 1980s paved the way for cloning of several new GPCRs, and the situation was quickly reversed (Dixon et al., 1986; Nathans & Hogness, 1983; reviewed in Civelli et al., 2013). Moreover, the sequencing of the human genome at the beginning of the current century has further increased the number of orphan GPCRs (Davenport et al., 2013). A method called reverse pharmacology represents a classical assay to identify ligands for orphan GPCRs (Civelli et al., 2013; Ngo et al., 2016; Yoshida, Miyazato, & Kangawa, 2012). The experimental approach involves expression of the orphan GPCR in a eukaryotic cellular system and screening of candidate ligands in signal transduction assays. This method has been successfully utilized to pair numerous GPCRs with their cognate ligands, but the number of deorphanizations

41 has decreased during the past years (Civelli et al., 2013; Ngo et al., 2016). More recently, several pharmacological and structure-based strategies have been developed to investigate the signaling mechanisms of orphan GPCRs (Hauser, Gloriam, Brauner-Osborne, & Foster, 2020; Laschet et al., 2018; Ngo et al., 2016). An alternative approach to assess the function of orphan GPCRs is to explore the phenotypes of animals, in which an is overexpressed or silenced (Davenport et al., 2013; Laschet et al., 2018). Nevertheless, pharmacological characterization of a GPCR does not necessarily require identification of an endogenous ligand, and some orphan receptors have even entered clinical trials as potential drug targets while their natural ligands are not known (Hauser, Attwood, Rask-Andersen, Schioth, & Gloriam, 2017; Laschet et al., 2018). This has been made possible by the concept of surrogate ligands, synthetic compounds that can bind to a receptor and initiate downstream signaling responses or modulate receptor activity (Ahmad, Wojciech, & Jockers, 2015). For example, Class A receptors GPR35, GPR55 and GPR84 have gained interest as potential pharmacological targets although they lack confirmed endogenous ligands (Hauser et al., 2017). Although the main emphasis on orphan GPCR research has been on ligand discovery, orphan receptors can also exert ligand-independent functions (Ahmad et al., 2015). Some receptors display a significant degree of constitutive activity, and consequently, trigger signaling responses in the absence of ligands (Seifert & Wenzel-Seifert, 2002). Orphan receptors can also heterodimerize with non-orphan GPCRs or form multimeric complexes with members of other protein families, thus modulating the activities of their interacting partners. In GPCR heterodimers, orphan receptors typically couple with non-orphan GPCRs that are members of the same receptor subfamily (Ahmad et al., 2015; Levoye, Dam, Ayoub, Guillaume, &

Jockers, 2006). For instance, GABAB receptors form a functional dimer, in which the GABAB1 receptor binds the ligand and the GABAB2 receptor facilitates G protein coupling and activation (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998). Additionally, orphan GPCRs in heterodimers may be subject to allosteric modulation even though they cannot bind orthosteric ligands (Levoye et al., 2006). While it is possible that some of the ligand-independent functions associated with orphan GPCRs result from the presence of a yet unidentified endogenous agonist under physiological conditions, it seems likely that some GPCRs are genuinely orphan without an existing endogenous ligand. Moreover, some receptors with identified ligands may act as conditional orphans behaving similarly to orphan GPCRs in the absence of the ligand (Ahmad et al., 2015; Levoye et al., 2006).

42 2.2 GPR37

GPR37 is an orphan GPCR that has been associated with certain neurological diseases and important physiological pathways in the brain (Berger, Acebron, Herbst, Koch, & Niehrs, 2017; B. M. Smith et al., 2017; N. J. Smith, 2015; Yang et al., 2016). However, the mechanisms of GPR37 function are still poorly characterized. The receptor was first cloned in 1997 from human brain, and the GPR37 gene was mapped to the long arm of 7 at position 7q31 (Marazziti et al., 1997; Zeng, Su, Kyaw, & Li, 1997). Within the brain, high expression of GPR37 mRNA has been described in putamen, medulla, caudate nucleus, corpus callosum, cerebellum, substantia nigra and hippocampus (Donohue et al., 1998; Marazziti et al., 1997; R. Takahashi & Imai, 2003; Yang et al., 2016; Zeng et al., 1997). Reports on receptor expression in other tissue types have varied. However, some expression in the liver, spinal cord, kidney, testis and placenta has been described (Donohue et al., 1998; Marazziti, Gallo, Golini, Matteoni, & Tocchini-Valentini, 1998; Marazziti et al., 1997; Zeng et al., 1997). At the cellular level, GPR37 can be found in both neuronal and glial cells and it is particularly enriched in oligodendrocytes (Cahoy et al., 2008; Imai et al., 2001; Yang et al., 2016).

2.2.1 Phylogenetic classification

GPR37 is a member of the β-subgroup of Class A/ Rhodopsin receptor family in the classification system described in chapter 2.1.1 (Fig. 5) (Fredriksson et al., 2003). Members of this relatively small subgroup bind peptide ligands (Fredriksson et al., 2003). GPR37 shows some sequence similarity with most of the family members, which is evident especially in the TM regions (Marazziti et al., 1998; Marazziti et al., 1997; Zeng et al., 1997). The closest homolog, GPR37 like 1 (GPR37L1), was identified shortly after GPR37 and named based on the sequence similarity between the two receptors (N. J. Smith, 2015; Valdenaire et al., 1998). The cDNA sequence identity between human GPR37 and GPR37L1 is 48% (Valdenaire et al., 1998). GPR37 has also been identified as a close homolog to endothelin and bombesin receptors (Donohue et al., 1998; Valdenaire et al., 1998). Especially the endothelin receptors are closely related to GPR37, and GPR37 has therefore gained the alternative name endothelin B receptor-like protein 1 (Fredriksson et al., 2003). The human GPR37 has been reported to display about

25% overall amino acid identity with human orthologs of ETBR and the bombesin

43 receptors neuromedin-B receptor and gastrin-releasing peptide receptor (Marazziti et al., 1997; Zeng et al., 1997). However, the sequence identity is higher in the TM domains, especially between the human ETBR and GPR37 that show 46% identity in these regions. On the other hand, the N-terminal regions of GPR37, GPR37L1 and endothelin receptors show little sequence identity (Fig. 6). The N-terminal domain of GPR37 is also significantly longer than the corresponding domains in its closest homologs (N. J. Smith, 2015). Moreover, there is some variation in the genomic structure of these closely related receptors. While the receptor coding sequence of endothelin and bombesin receptors is interrupted by several introns (Arai et al., 1993; Corjay et al., 1991), GPR37 and GPR37L1 carry only one intron interrupting the receptor-coding sequence within TM3 (Marazziti et al. 2001).

Fig. 5. Phylogenetic relationship between GPR37 and other β-cluster Class A receptors. GPR37 is shown in bold. Abbreviations of the closest homologs to GPR37:

BB1/BB2/BB3, bombesin BB1/2/3 receptor; BRS3, subtype-3;

ETAR/ETBR, endothelin A/B receptor; GPR37L1, G protein-coupled receptor 37 like 1; GRPR, gastrin-releasing peptide receptor; NMBR, neuromedin-B receptor. Modified from Fredriksson et al., 2003.

44 Fig. 6. Sequence alignment of the N-terminal domains of human GPR37, its mouse and rat orthologs and close homologs GPR37L1 and the endothelin receptors ETBR and

ETAR. The alignment was performed using Clustal Omega Multiple Sequence Alignment tool (https://www.ebi.ac.uk/Tools/msa/clustalo/) and formatted manually. The amino acids that are identical with the human GPR37 are highlighted in grey. The N-terminal signal peptide is shown in purple, the predicted first transmembrane domain in light blue and the putative N-glycosylation sites in dark blue. The sequences were retrieved from the UniProtKB database (http://www.uniprot.org). Accession codes: human GPR37, O15354; mouse GPR37, Q9QY42; rat GPR37, Q9QYC6; human GPR37L1, O60883; human ETAR, P25101; human ETBR, P24530.

2.2.2 Structural features

Since the crystal structure of GPR37 has not been resolved, the structural analysis of the receptor relies strongly on the investigation of secondary structural elements and sequence comparison between GPR37 and more extensively characterized

GPCRs. Presently, ETBR is the closest homolog to GPR37 with existing crystal structures (Shihoya et al., 2016; Shihoya et al., 2017). Although several orthologs

45 of GPR37 have been predicted through genomic studies, the mouse (m) and human (h) receptors are studied most extensively. The amino acid sequence and predicted structural features of hGPR37 are presented in Figure 7. The hGPR37 cDNA codes for a 613-amino acid protein and the mouse ortholog of the receptor is slightly smaller in size with 600 residues (Marazziti et al., 1998; Marazziti et al., 1997). The overall sequence identity between the human and mouse GPR37 is 83%. Most variation between the two proteins is observed in their N-terminal domains, while the TM regions and connecting loops are 98% identical (Marazziti et al., 1998). The N-terminal domain of GPR37 is exceptionally large for a rhodopsin-type GPCR. The predicted size of the hGPR37 N-terminus is about 260 amino acids (Fig 6 and 7) (Marazziti et al., 1997; Zeng et al., 1997) and that of mGPR37 about 250 residues (Fig 6) (Marazziti et al., 1998). Interestingly, there is some experimental evidence implying that the cell surface expression of GPR37 is increased when this domain is truncated (Dunham et al., 2009). Therefore, the N-terminus of GPR37 may relate to trafficking and function of the receptor. The N-terminal sequence of GPR37 begins with a hydrophobic signal peptide that targets the receptor to the ER. Additionally, the N-terminal domain contains several potential sites for N- and O- linked glycosylation (Marazziti et al., 1998; Marazziti et al., 1997; Zeng et al., 1997). In the hGPR37, the first putative N-glycosylation site is located downstream of the signal peptide at Asn36 and the other two sites are at Asn222 and Asn239 closer to the first TM domain (Fig. 6 and 7) (Marazziti et al. 1997). Additionally, the N-terminal domain of GPR37 is modified by O-glycosylation, although the specific sites have not been identified (Steentoft et al., 2013). Furthermore, the extracellular domains of GPR37 encompass cysteine residues that may form disulfide bonds (Fig 7). Two cysteines, one located at the border of TM3 and ECL1 and another within ECL2, are conserved among GPCRs and are likely to form a stabilizing intramolecular disulfide bridge. Additionally, the N-terminal tail contains two cysteines close to the putative N-glycosylation site at Asn36 (Marazziti et al., 1998). The C-terminal domain of GPR37 contains a cysteine-rich region immediately downstream of TM7 (Fig. 7). Similar cysteine-rich clusters are rare in GPCRs, although some receptors that are closely related to GPR37 also carry several cysteines in the C-terminal tail (Marazziti et al., 1998; Marazziti et al., 1997; Valdenaire et al., 1998). These cysteines might be modified by palmitoylation, as similar modifications have been demonstrated for many other GPCRs (Qanbar & Bouvier, 2003). One study also indicates that the cysteine cluster in GPR37 is important for receptor trafficking to the plasma membrane and may be involved in

46 the functional regulation of the receptor (Gandia et al. 2013). In addition to the cysteine-rich region, the intracellular domains of GPR37 carry several serine and threonine residues that could be modified by phosphorylation to facilitate receptor endocytic trafficking and downregulation (Marazziti et al., 1998; Marazziti et al., 1997). Furthermore, the GPR37 C-terminus encompasses a PDZ domain-binding motif (Gly-Thr-His-Cys) that could regulate receptor trafficking and signaling through interactions with PDZ domain-containing scaffold proteins (Fig. 7) (Dunham et al., 2009). Recent studies have identified a few proteins that interact with GPR37 via this domain (Dunham et al., 2009; Dutta, O'Connell, Ozkan, Sailer, & Dev, 2014; Tanabe, Fujita-Jimbo, Momoi, & Momoi, 2015). In 2009, syntenin- 1 was identified as a scaffold protein that interacts with GPR37 and enhances plasma membrane trafficking of the receptor (Dunham et al., 2009). In contrast to the effect of syntenin-1, interaction between GPR37 and the protein interacting with C-kinase (PICK1) was reported to reduce the expression levels of GPR37 (Dutta et al., 2014). Furthermore, an interaction between GPR37 and a complex of contactin-associated protein-like 2 (CASPR2) and multiple PDZ domain protein 1 (MUPP1) has been demonstrated (Tanabe et al., 2015). The TM domains of GPR37 contain certain amino acid residues that are highly conserved among Class A GPCRs and contribute to the stability and functional activity of these receptors. These residues include Asn2791.50, Asp3072.50, Trp3874.50, Pro4545.50, Pro5076.50 and Pro5467.50 [the numbers in the superscript refer to the Ballesteros-Weinstein numbering of the amino acid residues in GPCRs (Ballesteros & Weinstein, 1995)] in the TM1, TM2, TM4, TM5, TM6 and TM7 in the human receptor (Fig. 7) (Marazziti et al., 1997). Corresponding residues are also found in the mouse receptor (Marazziti et al., 1998). On the other hand, GPR37 does not have the main functionally relevant structural motifs that are conserved within Class A receptor family. In particular, the highly conserved tryptophan of the Cys-Trp-x-Pro motif in TM6 and the Asn-Pro-x-x-Tyr motif at the cytoplasmic end of TM7 are absent in GPR37. The tyrosine of the Asp-Arg-Tyr motif at the border of TM3 and ICL2 is also replaced by phenylalanine (Marazziti et al., 1998; Zeng et al., 1997).

47 Fig. 7. Snake plot presentation of the human GPR37. The diagram is based on the receptor cDNA sequence (Marazziti et al. 1997) and predicted topological regions presented in the UniProtKB/Swiss-Prot database (http://www.uniprot.org, accession code O15354). The amino acid sequence and main structural characteristics are shown in the figure. The N-terminal domain of GPR37 contains three putative N-glycosylation sites (green glycan structures) and several potential sites for O-glycosylation (pink) that were predicted with the NetOGlyc 4.0 server (http://www.cbs.dtu.dk/services/NetOGlyc/) (Steentoft et al., 2013). The cleavable signal peptide sequence is shaded with light blue. The cysteine residues that are likely to form disulfide bonds in the extracellular regions and the cysteine cluster in the receptor C-terminus are shown in purple. The residues that are most conserved in the TM domains of Class A GPCRs, the Asp-Arg-Phe motif (where tyrosine is replaced by phenylalanine) at the border of TM3 and ICL2 and the C- terminal PDZ-domain-binding motif are highlighted in blue.

48 2.2.3 Function

As GPR37 is an orphan receptor, relatively little is known about its signaling pathways. Although several attempts have been made to deorphanize GPR37, the results have been controversial at best. Initial efforts to identify the natural ligand of GPR37 focused on the agonists known to activate closely related endothelin and bombesin receptors (Marazziti et al., 1998; Marazziti et al., 1997; Zeng et al., 1997). However, these peptides failed to generate any signaling response on GPR37. Later on, four proteins, an invertebrate peptide head activator (HA), bioactive lipid metabolite neuroprotectin D1 (NPD1), regenerating islet-derived family member 4 (Reg4) and , have been suggested to act as endogenous ligands of GPR37 (Bang et al., 2018; Meyer, Giddens, Schaefer, & Hall, 2013; Rezgaoui et al., 2006; H. Wang et al., 2016). Especially prosaposin and the prosaposin-derived peptide fragment prosaptide have gained a lot of interest in the field as they are known to mediate neuroprotective and glioprotective effects (Campana, Hiraiwa, & O'Brien, 1998; N. Li, Sarojini, An, & Wang, 2010; O'Brien, Carson, Seo, Hiraiwa, & Kishimoto, 1994). In 2013, Meyer et al. reported that prosaposin and prosaptide promote internalization of GPR37, stimulate specific signaling in cells expressing GPR37 and protect primary astrocytes against oxidative stress (Meyer et al., 2013). Shortly afterwards, prosaposin was also found to increase GPR37 cell surface density and association with lipid rafts (Lundius et al., 2014). Nonetheless, the claim that these proteins are bona fide ligands of GPR37 has not been received without controversy. Research groups have reported inconsistent results when trying to confirm the findings (Nguyen et al., 2020; Yang et al., 2016) and direct evidence for receptor-ligand binding is still missing. Thus, none of the suggested ligands appears to meet the IUPHAR recommendations for receptor deorphanization described in chapter 2.1.5 (Davenport et al., 2013). A couple of studies have implied that GPR37 might convey downstream signaling via the inhibitory Gαi-mediated pathway (Meyer et al., 2013; Zheng et al., 2018). Nevertheless, in the absence of a known activating ligand, these reports have relied on methods that do not measure direct receptor-ligand binding and the actual downstream signaling mechanism of GPR37 remains to be resolved. Additionally, GPR37 has been identified as an interacting partner for several other GPCRs. These receptors include D2R, adenosine A2A receptor (A2AR), 5-hydroxytryptamine receptor 4, β2-adrenergic receptor and glucagon-like peptide 1 receptor (Dunham et al., 2009; Hertz, Terenius, Vukojevic, & Svenningsson, 2019; Huang et al., 2013; Kittanakom et al., 2014; Morato et al., 2017; Sokolina et al., 2017). The interactions

49 between GPR37 and D2R, A2AR and 5-hydroxytryptamine receptor 4 have also been confirmed in cell biological assays (Dunham et al., 2009; Hertz et al., 2019; Morato et al., 2017; Sokolina et al., 2017). However, the functional interplay between these GPCRs has not yet been characterized carefully. The physiological relevance of GPR37 has been more intensively researched, and several studies have taken advantage of GPR37 knock-out mice in the absence of the endogenous ligand (Marazziti et al., 2004; B. M. Smith et al., 2017; X. Zhang et al., 2020). To date, GPR37 has been linked to oligodendrocyte differentiation and myelination (B. M. Smith et al., 2017; Yang et al., 2016) along with certain pathological conditions that are described below in more detail. Other biological functions associated with GPR37 include Wnt/β-catenin signaling in neurogenesis (Berger et al., 2017), modulation of cell migration during development (Saadi, Shan, Marazziti, & Wray, 2019), Sertoli cell maturation and proliferation (La Sala et al., 2015) and protective signaling pathways activated following cerebral ischemia and inflammation (Bang et al., 2018; McCrary et al., 2019).

Parkinson’s disease

Parkinson’s disease is a neurodegenerative disorder characterized by motor and non-motor symptoms resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta (Kalia & Lang, 2015). GPR37 was originally linked to Parkinson’s disease in 2001 when it was identified as an interacting partner of the E3 ubiquitin ligase (Imai et al., 2001). In 1998, Kitada and colleagues reported that mutations in the PA R K2 gene encoding for Parkin cause autosomal recessive juvenile parkinsonism (AR-JP), an early onset familial form of Parkinson’s disease (Kitada et al., 1998). In a subsequent study, Imai and his colleagues aimed to identify novel substrates for Parkin and isolated GPR37 from a human brain library in a yeast two-hybrid screen using Parkin as a bait (Imai et al., 2001). Thus, GPR37 acquired an alternative name Parkin-associated -like receptor. The authors also reported accumulation of an insoluble form of GPR37 in cellular models overexpressing the receptor and in the brains of AR- JP patients (Imai et al., 2001). Later on, GPR37 aggregates were found also in the core of Lewy bodies in brain sections from patients with idiopathic Parkinson’s disease (Murakami et al., 2004). Furthermore, studies combining GPR37 overexpression with Parkin deletion in mice indicated that this combination leads to neurotoxic effects (Kitao et al., 2007; H. Q. Wang et al., 2008). Intracellular aggregation of unfolded GPR37 and impaired ubiquitination of the receptor by

50 Parkin were therefore suggested to cause cytotoxicity and loss of dopaminergic neurons typical for Parkinson’s disease pathogenesis (Imai et al., 2001; Kitao et al., 2007; H. Q. Wang et al., 2008). However, this original view of GPR37 as a substrate of Parkin in the cytosolic proteasomal degradation pathway has been challenged by more recent research on Parkin function. Numerous studies indicate that Parkin functions together with the serine/threonine kinase PTEN-induced kinase 1 (PINK1) in mitochondria to promote degradation of damaged mitochondria (Seirafi, Kozlov, & Gehring, 2015). Furthermore, crystallographic studies on Parkin structure have revealed that the activity of the enzyme is regulated by several mechanisms of autoinhibition, and the recruitment to mitochondria by PINK1 is required for Parkin activation (Riley et al., 2013; Seirafi et al., 2015; Trempe et al., 2013; Wauer & Komander, 2013). Thus, it remains unclear how Parkin might be activated to facilitate degradation of GPR37 and other identified cytosolic substrate proteins. Despite the controversy related to the proposed functional connection between GPR37 and Parkin, several studies link GPR37 to the signaling pathways associated with Parkinson’s disease. Research with GPR37 knock-out and transgenic animals implies that GPR37 plays a role in the modulation of dopaminergic neurotransmission (Imai et al., 2007; Marazziti et al., 2011; Marazziti et al., 2004; Marazziti et al., 2007; X. Zhang et al., 2020). GPR37 knock-out mice have also been reported to display altered response to the psychostimulants amphetamine and cocaine that elevate dopamine levels, although the results provided by individual research groups are not entirely consistent with each other (Imai et al., 2007; Marazziti et al., 2011; Marazziti et al., 2004; Marazziti et al., 2007). Moreover, direct interaction between GPR37 and proteins involved in dopaminergic signaling and Parkinson’s disease has been reported. In one study, GPR37 was shown to associate with the dopamine transporter and deletion of GPR37 in mice led to increased cell surface levels and activity of the transporter (Marazziti et al., 2007). Correspondingly, the dopamine transporter cell surface expression and activity have been reported to decrease upon coexpression with GPR37 in human embryonic kidney (HEK) 293 cells (Benleulmi-Chaachoua et al.,

2018). GPR37 interacts also with D2R and A2AR, two GPCRs involved in signaling pathways relevant for Parkinson’s disease (Dunham et al., 2009; Hertz et al., 2019; Morato et al., 2017; Sokolina et al., 2017). However, very little is known about the functional consequences of these interactions. Both D2R and A2AR have been reported to increase the cell surface expression of GPR37 (Dunham et al., 2009; Sokolina et al., 2017). On the other hand, GPR37 appears to negatively regulate

A2AR cell surface expression and function (Morato et al., 2019; Morato et al., 2017).

51 Association with other diseases

In addition to Parkinson’s disease, GPR37 has been implicated in some neuropsychiatric disorders that involve dysfunctions in the dopaminergic system (Fujita-Jimbo et al., 2012; Tanabe et al., 2015; Tomita et al., 2013). Microarray gene expression analyses of post-mortem human brain tissue indicate that GPR37 expression is decreased in the brains of major depressive disorder patients (Aston, Jiang, & Sokolov, 2005; Tomita et al., 2013). In contrast, GPR37 expression has been reported to be increased in the brains of bipolar disorder patients (Tomita et al., 2013). Additionally, GPR37 has been associated with autism spectrum disorder (Fujita-Jimbo et al., 2012). The chromosomal location of GPR37 at 7q31 coincides with the locus called AUTS1 that has been linked to autism (Fujita-Jimbo et al., 2012; Marazziti et al., 1997). Furthermore, two mutations in the receptor, deletion of Phe312 in TM2 and substitution Arg558Gln in the C-terminal tail, have been identified in affected individuals (Fujita-Jimbo et al., 2012). However, both mutations were also found in unaffected family members, suggesting that these mutations alone are not sufficient to cause the disease (Fujita-Jimbo et al., 2012). More recently, the authors reported that the GPR37 Arg558Gln mutation affects the interaction between GPR37 and PDZ domain-containing proteins contactin- associated protein-like 2 and multiple PDZ domain protein 1 resulting in impaired trafficking of the receptor to the cell surface (Tanabe et al., 2015). The recently discovered connection between GPR37 and myelination (discussed in more detail below) also implies that the receptor may be involved in diseases associated with impaired myelination or demyelination (B. M. Smith et al., 2017; Yang et al., 2016). Moreover, a few studies have suggested a possible link between GPR37 and certain types of cancer (Huang et al., 2014; F. Liu et al., 2014; Reichl et al., 2020; Toyota et al., 2001; H. Wang et al., 2016). Nevertheless, most of these studies reported only differential expression or modification of GPR37 in cancer, while the functional involvement of GPR37 in the disease was not explored.

Oligodendrocyte differentiation and myelination

Recent research indicates that GPR37 is also involved in oligodendrocyte differentiation and myelination (B. M. Smith et al., 2017; Yang et al., 2016). A few years ago, Yang. et al. (2016) reported that GPR37 is enriched in pre-myelinating and myelinating oligodendrocytes, a finding that is supported by previous analyses of gene expression in glial cells (Cahoy et al., 2008; Y. Zhang et al., 2014). The

52 authors also discovered that genetic deletion of GPR37 in mice resulted in accelerated differentiation of oligodendrocytes and hypermyelination (Yang et al. 2016). Based on these findings, they concluded that GPR37 acts as a negative regulator of oligodendrocyte differentiation during the transition from early- differentiated to mature oligodendrocytes. Furthermore, this inhibitory effect appeared to be mediated by suppression of the ERK1/2 signaling pathway that has been associated with regulation of myelin thickness (Yang et al., 2016). Shortly afterwards, the research group of Randy Hall reported that GPR37 interacts with myelin-associated glycoprotein (MAG) in cells, and GPR37 deletion in mice reduces the brain expression of MAG (B. M. Smith et al., 2017). Moreover, loss of GPR37 was found to increase susceptibility to demyelination induced by cuprizone, a copper-chelating agent used to cause toxic demyelination in rodents (B. M. Smith et al., 2017). Interestingly, transcriptional profiling of gene expression in a major depressive disorder has revealed that in addition to GPR37, the expression of several involved in oligodendrocyte function and myelination is decreased in the brains of affected individuals (Aston et al., 2005). Besides, MAG was among the genes that were reported to show significantly lower expression in the disease (Aston et al., 2005). Collectively, these findings provide promising evidence for a role of GPR37 in myelination, although more research is needed to establish a proper connection between the receptor and the mechanism of myelin formation.

2.3 Proteolytic processing

Proteolysis is an irreversible posttranslational event that can regulate the biological function of proteins in multiple ways. In addition to degradative pathways, such as ERAD and lysosomal degradation, proteolysis can occur in a more precise, targeted manner that is limited to the cleavage of specific peptide bonds. This kind of limited proteolysis, also known as proteolytic processing, may involve, for example, signal peptide removal, activation or inactivation of proteins by proteolytic cleavage or modulation of signaling and biochemical pathways (Klein, Eckhard, Dufour, Solis, & Overall, 2018; Overall & Blobel, 2007). Proteolytic processing is mediated by protease enzymes that hydrolyze peptide bonds between amino acids of the protein polypeptide chain. Mammalian proteases can be divided into five main catalytic classes: aspartyl-, cysteine-, metallo-, serine- and threonine proteases (Klein et al., 2018; Lopez-Otin & Bond, 2008). Of these, metalloproteases form the largest group that includes both secreted and membrane-bound enzymes (Bond, 2019). The activities of protease enzymes are regulated at various levels. For instance,

53 proteolytic cleavage depends on the tissue, cell type and intracellular localization of the enzyme and the substrate, activation and inactivation of the enzyme and communication with various interacting proteins (Turk, Turk, & Turk, 2012). This section covers the main proteases and proteolytic mechanisms relevant for my thesis.

2.3.1 Proprotein convertases

Proprotein convertases are serine proteases that use an active site serine to hydrolyze peptide bonds. As the name of the protease family implies, these proteases convert immature proproteins into their biologically active, or in some cases inactive, forms by cleaving their prodomain sequences. Proprotein convertases are involved in many biologically important processes and their substrates range from hormones, receptors, cytokines and growth factors to other protease enzymes (Seidah & Prat, 2012). Out of nine proprotein convertase enzymes, seven cleave their substrates after basic amino acids at the preferred (Arg/Lys)-2nx-Arg/Lys↓ (where n = 0–3 amino acids and x represents any amino acid) cleavage site (Seidah, Sadr, Chretien, & Mbikay, 2013; Turpeinen, Ortutay, & Pesu, 2013). The remaining two enzymes are involved in cholesterol and/or lipid homeostasis and cleave their substrates after nonbasic residues or use an indirect mechanism to promote degradation of cell surface proteins (Seidah & Prat, 2012). All proprotein convertases contain a signal peptide that is cleaved in the ER and a prodomain that mediates the folding and activation of the enzyme (Fig. 7A). To become biologically active, proprotein convertases undergo autocatalytic cleavage of the prodomain that precedes the catalytic domain. The domain architecture of the C-terminal part varies, and the enzymes may contain a transmembrane segment or exist as soluble proteins (Seidah & Prat, 2012; Turpeinen et al., 2013).

Furin

Furin was the first identified proprotein convertase enzyme (van de Ven et al., 1990). It is a single-pass membrane protein (Fig. 8A) that cycles between the TGN, cell surface and the vesicles of the endosomal pathway (Molloy, Thomas, VanSlyke, Stenberg, & Thomas, 1994; Thomas, 2002). Additionally, the extracellular part of furin can be released to the extracellular space after proteolytic cleavage of the enzyme (Vey, Schafer, Berghofer, Klenk, & Garten, 1994; Vidricaire, Denault, & Leduc, 1993). Furin is also ubiquitously expressed in all mammalian tissues and

54 cell types (Seidah & Prat, 2012). Therefore, this multifaceted enzyme can cleave its substrate proteins in various cellular locations. Furin uses the canonical polybasic proprotein convertase substrate cleavage site. The substrate selectivity is additionally determined by amino acids close to the cleavage site and secondary and tertiary structures of the substrate proteins (Henrich, Lindberg, Bode, & Than, 2005; Shiryaev et al., 2013; Turpeinen et al., 2011). Although furin has certain unique substrates, it also shares some redundant or complementary functions with other proprotein convertases (Seidah et al., 2013; Turpeinen et al., 2013).

2.3.2 ADAM family metalloproteases

A Disintegrin And Metalloprotease (ADAM) enzymes are single-spanning transmembrane proteins that belong to the metzincin family of metalloproteases. These enzymes use zinc metal ion for their catalytic mechanism (Seals & Courtneidge, 2003). Together with matrix metalloproteases (MMPs), ADAMs comprise one of the most intensively studied subgroups of metalloproteases (Klein et al., 2018). However, only about half of the ADAMs contain a zinc-binding catalytic site consensus motif (His-Glu-x-x-His, where X is any amino acid), whereas some of the ADAM family members are not catalytically active metalloproteases (Becherer & Blobel, 2003). ADAMs possess a modular domain organization and require signal peptide and prodomain removal to become biologically active. The prodomain cleavage is mediated by furin or other members of the proprotein convertase family (Weber & Saftig, 2012). To date, there are more than 20 ADAM family members found in humans (Wetzel, Seipold, & Saftig, 2017). These enzymes regulate various biological processes including signaling, development, cell adhesion and migration, as well as proteolytic processing of pathologically relevant substrates (Hsia et al., 2019; Reiss & Saftig, 2009). Typical ADAM substrates are single-spanning type I or II membrane proteins or glycosylphosphatidylinositol-anchored proteins. Although ADAMs have been implicated in the processing of a wide range of substrate proteins, it has remained unclear if their physiological substrates include multi- spanning transmembrane proteins (Weber & Saftig, 2012). However, the metalloprotease domains of ADAM10 and ADAM17 can be cleaved by other metalloproteases (Scharfenberg et al., 2020; Tousseyn et al., 2009), and a recent report implies that the secreted metalloprotease domains are able to process soluble substrate proteins (Scharfenberg et al., 2020). Thus, these proteases are likely to function in a more versatile manner than previously anticipated.

55 ADAM10

ADAM10, together with ADAM17, is the most extensively characterized ADAM family member. It has a wide range of substrate proteins including N- and E- cadherin, ephrin receptors, pro-epidermal growth factor and Notch (Reiss & Saftig, 2009; Weber & Saftig, 2012). It is also involved in certain pathological conditions as its substrates include disease-associated proteins such as cellular prion protein and amyloid precursor protein (Kuhn et al., 2010; Lammich et al., 1999; Vincent et al., 2001). ADAM10 plays a particularly important role in brain development and physiology (Saftig & Lichtenthaler, 2015). It is widely expressed and can be found ubiquitously in all major cell types in the brain (Karkkainen, Rybnikova, Pelto- Huikko, & Huovila, 2000; Sharma et al., 2015). The enzyme can cleave substrate proteins in a constitutive manner, but its activity can also be upregulated by certain stimuli (Maretzky et al., 2015). The mechanisms that govern the substrate selectivity of ADAM10 are incompletely understood. ADAM10 does not recognize a specific cleavage site motif, but it appears to favor certain amino acids close to the cleavage site (Caescu, Jeschke, & Turk, 2009; Tucher et al., 2014). Substrate cleavage depends on the distance between the cleavage site and the TM region (Matthews, Szyroka, Collier, Noy, & Tomlinson, 2017). Additionally, the cleavage site accessibility is determined by the substrate conformation that is typically tightly regulated (M. Hartmann, Herrlich, & Herrlich, 2013; M. Hartmann et al., 2015; Linneberg, Toft, Kjaer-Sorensen, & Laursen, 2019). ADAM10 itself is also regulated at multiple levels. Like other ADAM family members, the extracellular part of ADAM10 consists of distinct domains (Fig. 8B). The disintegrin and cysteine-rich domains prevent unrestricted substrate accessibility by masking the catalytic metalloprotease domain (Fig. 8C) (Seegar et al., 2017). On the other hand, these domains may also interact with the substrate and direct the protease domain for effective cleavage after conformational changes in the enzyme (Janes et al., 2005; Seegar et al., 2017). The activity of ADAM10 is also regulated by the membrane lipid composition and interactions with other proteins (Bleibaum et al., 2019; M. Hartmann et al., 2013). Especially the TspanC8 subgroup of tetraspanins play an important role controlling ADAM10 trafficking and function (Matthews et al., 2017; Saint-Pol et al., 2017). Interaction with different TspanC8 members may also enable ADAM10 to adopt distinct conformations and position the metalloprotease domain in a favorable orientation to cleave specific substrates (Matthews et al., 2017).

56

Fig. 8. Domain organization of furin and ADAM10. Both furin (A) and ADAM10 (B) have an extracellular multidomain structure that is followed by a transmembrane (TM) segment and a cytoplasmic domain. The enzymes are converted into their catalytically active forms after proteolytic removal of the signal peptide in the ER and the prodomain during protein maturation. Modified from Lambrecht, Vanderkerken, & Hammad, 2018 and Turpeinen et al., 2013 C) Crystal structure of the mature human ADAM10 ectodomain (created using Mol* at RCSB Protein Data Bank, structure ID: 6BE6) (Seegar et al., 2017). The cysteine-rich domain partially occludes the active site of the enzymes and prevents unrestricted substrate access. CT/ NT, C-/ N-terminal end of the protein structure.

57 2.3.3 Ectodomain shedding

Ectodomain shedding is a proteolytic process involved in the regulation of several membrane proteins. During the shedding process, the membrane protein substrate undergoes proteolytic cleavage adjacent to or within its membrane-anchoring domain, resulting in the release of a significant portion of the extracellular ectodomain (Clark, 2014; Lichtenthaler, Lemberg, & Fluhrer, 2018). Ectodomain shedding affects almost all functional categories of membrane proteins and is therefore involved in a wide range of cellular mechanisms and several pathological conditions (Hayashida, Bartlett, Chen, & Park, 2010). Proteins can be activated or inactivated by ectodomain shedding, or it can be a mechanism to control protein turnover. For instance, shedding contributes to the release of membrane-bound cytokines and growth factors and controls the abundance of surface receptors and cell adhesion molecules (Lichtenthaler et al., 2018). In some cases, ectodomain shedding is followed by additional proteolytic cleavage(s) within the transmembrane domain of the substrate protein. This multi-step proteolytic process is called regulated intramembrane proteolysis and may lead to release of intracellular domain fragments that enter the nucleus and control gene transcription (Brown, Ye, Rawson, & Goldstein, 2000). The protease enzymes that trigger ectodomain shedding are collectively called sheddases. Enzymes belonging to diverse protease families have been shown to act as sheddases, but many of them also exhibit functions that are not related to ectodomain shedding (Clark, 2014; Lichtenthaler et al., 2018). The enzymes that cleave the substrate ectodomain a short distance from the membrane-tethered domain are typically themselves membrane-bound proteins. The ADAM-family metalloproteases are a typical example of such enzymes (Reiss & Saftig, 2009; Weber & Saftig, 2012). Especially ADAM10 and ADAM17 are well characterized sheddases with multiple physiologically relevant substrates (saftig & Reiss 2010). However, an increasing number of soluble sheddases and intramembrane proteases, which cleave their substrates within the transmembrane domain, are found to mediate ectodomain shedding (Lichtenthaler et al., 2018).

2.3.4 Proteolytic processing of GPCRs

Limited proteolysis has emerged as a mechanism involved in the functional regulation of certain GPCRs. Adhesion receptors constitute an exceptional family of GPCRs that contain a conserved proteolytic site in their N-terminal domains

58 (Arac et al., 2012). Although almost all Adhesion receptors are considered orphans, several family members can be activated by a tethered agonist encrypted in the receptor N-terminus (Purcell & Hall, 2018; Vizurraga et al., 2020). The mechanism involves autoproteolytic cleavage in the membrane proximal GAIN domain. Consequent dissociation of the N-terminal fragment exposes the agonist peptide in the remaining part of the N-terminal domain. However, not all adhesion family GPCRs are activated by this mechanism, and the receptors may also be stimulated by allosteric interaction with external ligands (Vizurraga et al., 2020). In the Class A/ Rhodopsin family, PARs exhibit a similar tethered agonist-mediated activation mechanism (Hamilton & Trejo, 2017). Nonetheless, PARs do not contain an autoproteolytic site, but instead are cleaved by various endogenous and exogenous proteases. While initial studies identified thrombin as the cleaving enzyme for PAR1, PAR3 and PAR4 and trypsin as the major PAR2 protease, many other proteases have been implicated in the processing of these receptors. Some of the proteases are involved in the activation mechanism of PARs, but others may render the receptor inactive (Heuberger & Schuepbach, 2019; Renesto et al., 1997). Receptor cleavage at distinct sites may also produce divergent tethered ligand peptides and lead to biased signaling (Hamilton & Trejo, 2017). Although the adhesion receptors and PARs have attracted most of the attention when it comes to proteolytic processing of GPCRs, some other receptors have also been demonstrated to undergo proteolytic cleavage (Table 3). Interestingly,

GPR37L1 and ETBR, two of the closest homologs to GPR37, are subjected to N- terminal cleavage (J. L. J. Coleman et al., 2020, Grantcharova et al. 2002). ETBR is cleaved in an agonist-dependent manner and proteolytic processing has been reported to alter the receptor function (Grantcharova et al. 2002). In contrast, GPR37L1 is an orphan GPCR and the functional consequences of receptor cleavage are not known (J. L. J. Coleman et al., 2020). In general, the physiological significance of the proteolytic processing of GPCRs has remained largely unexplored, and very little is known about the connection between GPCR signaling and limited proteolysis. The thyroid-stimulating hormone receptor (TSHR) is one of the most extensively studied GPCRs that undergoes proteolytic cleavage. The metalloprotease-mediated cleavage of the receptor is believed to occur in an activation-dependent manner, and it is followed by ectodomain shedding (Kaczur et al., 2007; Latif, Ando, & Davies, 2004). The β1-adrenergic receptor and V2 vasopressin receptor are also reported to be cleaved following agonist binding (Hakalahti et al., 2013; Hakalahti et al., 2010; Kojro & Fahrenholz, 1995). The cleavage of the parathyroid hormone receptor, in contrast, results in reduced protein

59 stability and is inhibited by agonist peptides (Klenk, Schulz, Calebiro, & Lohse,

2010). In the case of the α1D-adrenergic receptor, cleavage of the receptor N- terminus is required for proper plasma membrane localization and function (Kountz et al., 2016). More recently, the N-terminal cleavage of the sphingosine 1- phosphate receptor 2 was reported to result in constitutive receptor activity and ERK1/2 signaling (El Buri et al., 2018). Intriguingly, some adhesion family receptors can be N-terminally processed by proprotein convertases or metalloproteases in addition to the GAIN domain cleavage, although the functional outcomes are not clear (Cork et al., 2012; Fukuzawa & Hirose, 2006; Moriguchi et al., 2004). There are also examples of GPCRs that are cleaved in ECLs and intracellular domains. For instance, the cleavage of the Frizzled-2 receptor, angiotensin II type 1 receptor and GPR50 in the C-terminal tail leads to translocation of the C-terminal fragment to the nucleus where it might be able to modulate gene transcription (Ahmad et al., 2020; Cook, Mills, Naquin, Alam, & Re, 2007; Mathew et al., 2005).

Table 3. GPCRs that are subjected to limited proteolysis.

Receptor Cleavage site Enzyme type References Adhesion receptors N-terminus Autoproteolytic Vizurraga et al., 2020 Metalloprotease Cork et al., 2012 Proprotein convertase Fukuzawa & Hirose, 2006

α1DAR N-terminus Not known Kountz et al., 2016 AT1R N-terminus, C-terminus Not known Cook et al., 2007

β1AR N-terminus Metalloprotease Hakalahti et al., 2010

β2AR N-terminus Not known Jockers et al., 1999 C5aR N-terminus, ECL3 Metalloprotease van den Berg et al., 2012

ETBR N-terminus Metalloprotease Grantcharova et al., 2002 Fz2 C-terminus Not known Mathew et al., 2005 GPR37L1 N-terminus Metalloprotease Coleman et al., 2020 GPR50 C-terminus Not known Ahmad et al., 2020 GPR107a N-terminus Proprotein convertase Tafesse et al., 2014 PAR1-4 N-terminus Multiple enzymes Heuberger & Schuepbach, 2019 PTHR N-terminus Metalloprotease Klenk et al., 2010 S1PR2 N-terminus Metalloprotease El Buri et al., 2018 TSHR N-terminus Metalloprotease Couet et al., 1996

V2R TM2, ECL1 Not known Kojro & Fahrenholz, 1995 a putative GPCR. Abbreviations: α1DAR, α1D-adrenergic receptor; AT1R, angiotensin II type 1 receptor;

β1/2AR, β1/2-adrenergic receptor; C5aR, ; ETBR, endothelin B receptor; Fz2, frizzled-2; PAR1-4, protease-activated receptor 1-4; PTHR, parathyroid hormone receptor; TSHR, thyroid- stimulating hormone receptor; V2R, V2 vasopressin receptor

60 3 Aims

GPR37 is abundantly expressed in the central nervous system and has been implicated in several processes naturally occurring in the brain. The receptor has been linked to dopaminergic signaling, myelin formation as well as Wnt/β-catenin signaling. Moreover, GPR37 has been associated with Parkinson’s disease and various other neurological disorders. Therefore, it provides a promising drug target to treat neurological diseases. The activation- and regulatory mechanisms of GPR37, however, remain poorly understood. The overall aim of this project was to investigate the biosynthesis and processing of GPR37 in order to gain more insight into the mechanisms that could regulate receptor activity. That knowledge in turn will help to understand the functional role of GPR37 in health and disease and will make it possible to determine the full potential of GPR37 as a therapeutic target.

The specific aims of the research were: 1. To characterize the biosynthesis of GPR37 and to investigate the proteolytic mechanisms involved in the regulation of the receptor. 2. To identify the enzymes that cleave the N-terminal domain of GPR37 and to determine their cleavage sites and mechanisms of action. 3. To demonstrate that the N-terminal cleavage is a physiologically relevant process that takes place in native tissue.

61

62 4 Materials and methods

Various cell biological and biochemical methods were used in the present study. Figure 9 outlines the methods used starting from receptor expression and cell culture methods and ending with protein detection and data analysis.

Fig. 9. General outline of the methods used and their connections to each other.

63 4.1 Receptor expression

4.1.1 Animals

The wild-type and GPR37 knock-out mice in the C57BL/6J genetic background (Strain Name: B6.129P2-GPR37tm1Dgen/J) were acquired from the Jackson Laboratory (Bar Harbor, ME, USA). The study protocol was approved by the University of Barcelona Committee on Animal Use and Care (permit number: 7085). The desired brain regions (striatum, hippocampus and cerebellum) were collected from 8-week-old male mice. The animals were euthanized by cervical dislocation and the freshly harvested brain tissue was used for preparation of cellular membranes. The brain tissue from cortex, hippocampus and cerebellum of ADAM10 conditional knock-out mice (Adam10f/f; CamKIIα-Cre) and wild-type littermate controls (Adam10F/F or Adam10F/+) (Prox et al., 2013), harvested at postnatal day 20, were generously provided by Professor Paul Saftig (Kiel University, Kiel, Germany).

4.1.2 DNA constructs

The GPR37 cDNA constructs contained N- and/or C-terminal epitope tags which enable efficient purification and detection of the receptors with epitope-specific antibodies. The DNA constructs and their expression vectors are presented in Table 4. The hGPR37 and C-terminally FLAG-tagged mGPR37 constructs in pcDNA3 vector were kindly provided by Ryosuke Takahashi (Kyoto University, Japan). The hGPR37 was further modified to create two C-terminally FLAG (DYKDDDDK)- tagged constructs. A construct with a cleavable influenza hemagglutinin (HA) signal peptide (KTIIALSYIFCLVFA) and an N-terminal Myc epitope (EQKLISEEDL) was used in most of the experiments, whereas a construct containing endogenous signal peptide without an N-terminal epitope was created as an additional control. Both constructs were generated using the hGPR37- pcDNA3 as a template for polymerase chain reaction (PCR) amplification. The digested PCR products were then ligated into a modified pcDNA5/FTR/TO vector (Invitrogen) termed pFT-SMMF (Pietila et al., 2005). The Myc- and FLAG-tagged δ-opioid receptor construct, which was used as a control GPCR, was created in a similar manner (Leskelä, Markkanen, Pietilä, Tuusa, & Petäjä-Repo, 2007). The N- terminally NanoLuc- and HA-tagged hGPR37 construct was created by inserting the HA-tag sequence (YPYDVPDYA) into the pNLF1-secN vector (Promega)

64 using the QuikChange II site-directed mutagenesis kit (Agilent Technologies). The hGPR37 cDNA was then cloned into the pNLF1-secN-HA plasmid (Promega Madison). Site-directed mutagenesis was used to alter the nucleotide sequence of several cDNA constructs. Point mutations and short deletions were introduced to the Myc- hGPR37-FLAG pFT-SMMF template using the QuikChange Lightning mutagenesis kit (Agilent Technologies) according to the protocol provided by the manufacturer. The construct without the C-terminal FLAG epitope was created with the same technique by inserting a stop codon before the sequence encoding for the epitope tag. The hADAM10 E384A construct was generated using the HA- tagged hADAM10 in pEAK12 vector (provided by Professor Mikko Hiltunen, University of Eastern Finland, Kuopio, Finland) (Lichtenthaler et al., 2003) as a template. The desired mutations were verified by DNA sequencing. The oligonucleotide primers used for PCR amplification are listed in the original publications (I, Table S1 and II, Materials and methods). The deletion mutant constructs in Fig. 9 of this thesis were created using the oligonucleotide primers shown in Table 5 and the corresponding reverse complements.

Table 4. DNA constructs and expression vectors.

Construct Vector Study Myc-hGPR37-FLAG pFT-SMMF I, II Myc-hGPR37 pFT-SMMF I hGPR37-FLAG pFT-SMMF I Myc-hGPR37(E167D;Q168H)-FLAG pFT-SMMF I Myc-hGPR37(E167D;Q168N)-FLAG pFT-SMMF I Myc-hGPR37(ΔE167-Q168)-FLAG pFT-SMMF I Myc-hGPR37(ΔE166-Q169)-FLAG pFT-SMMF I Myc-hGPR37(R54A)-FLAG pFT-SMMF II Myc-hGPR37(R51;52;54A)-FLAG pFT-SMMF II Myc-hGPR37(ΔR51-R54)-FLAG pFT-SMMF II Myc-hGPR37(ΔR68-R73)-FLAG pFT-SMMF II Myc-hGPR37(ΔR121-R126)-FLAG pFT-SMMF II NanoLuc-HA-hGPR37 pNLF1-secN II mGPR37-FLAG pcDNA3 I, II Myc-hδOR-FLAGa pFT-SMMF I hADAM10-HAb pEAK12 II hADAM10(E384A)-HA pEAK12 II References: a Leskelä et al., 2007 b Lichtenthaler et al., 2003. Abbreviations: ADAM, a disintegrin and metalloprotease; h, human; m, mouse; OR, opioid receptor.

65

Table 5. Oligonucleotides for site-directed mutagenesis.

Template construct Mutations Oligonucleotide sequence Myc-hGPR37-FLAG Δ167-168 5’-CCGGGCGTAGCCAGAGTGTGAAGACAGTC-3’ Myc-hGPR37-FLAG Δ166-169 5’-CATTTCCGGGCGTAGCGTGAAGACAGTCCCC-3’ Myc-hGPR37(Δ166-169)-FLAG Δ164-171 5’-CTGGCATTTCCGGGACAGTCCCGGAGC-3’ Myc-hGPR37(Δ166-169)-FLAG Δ165-172 5’-CATTTCCGGGCGTGTCCCCGGAGCCAG-3’ Myc-hGPR37(Δ164-171)-FLAG Δ164-176 5’-GCGCTGGCATTTCCGGAGCGATCTTTTTTACTG-3’ Myc-hGPR37(Δ165-172)-FLAG Δ165-177 5’-CTGGCATTTCCGGGCGTGATCTTTTTTACTGGCC-3’ Myc-hGPR37(Δ165-177)-FLAG Δ165-181 5’-CGCTGGCATTTCCGGGCGTTGGCCAAGG-3’ Δ deletion

4.1.3 Cell lines

Various mammalian cell lines were used as models for receptor expression. The cell lines (Table 6) were maintained at 37 ˚C in a humidified environment of 5% CO2. The LoVo cells were cultured in Ham’s F-12 medium and the other cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM). The culture medium was supplemented with 10% fetal bovine serum (FBS) and the antibiotics as indicated in Table 6.

Table 6. Cell lines.

Cell line Phenotype Source Antibiotics Study Flp-In-HEK293 FRT-site Invitrogen PS, Zeoc. I

HEK293i Myc-hGPR37-FLAG Myc-hGPR37-FLAG (i) made in lab PS, Blast, Hyg. I, II HEK293S Sigma-Aldrich PS II HEK293Ta Prof. C. Garbens PS II HEK293T A10 -/-a ADAM10 knock-out Prof. C. Garbens PS II HEK293T A17 -/-a ADAM17 knock-out Prof. C. Garbens PS II LoVob no furin expression Sigma-Aldrich PS II MEFc Prof. P. Saftig PS II MEF A10 -/-c ADAM10 knock-out Prof. P. Saftig PS II SH-SY5Y Prof. M. Hiltunen PS I, II References: a Riethmueller et al., 2016 b S. Takahashi et al., 1995 c D. Hartmann et al., 2002. Abbreviations: ADAM, a disintegrin and metalloprotease; Blast., Blasticidin; FRT, flippase recognition target; HEK, human embryonic kidney; Hyg., Hygromycin; i, inducible; LoVo, human colon carcinoma cell line; MEF, mouse embryonic fibroblast; PS, Penicillin/Streptomycin; SH-SY5Y, human bone marrow neuroblastoma cell line.

66 A stable cell line that expresses the Myc- and FLAG-tagged hGPR37 in a tetracycline-inducible manner was commonly used in the experiments. The cell line was generated by co-transfecting inducible HEK293i cells (Apaja et al. 2006) with the Myc-hGPR37-FLAG receptor construct and pOG44 plasmid (Invitrogen) under hygromycin (400µg/ml, Invivogen) and blasticidin S (4µg/ml, Invivogen) selection using the Lipofectamine 2000 transfection reagent. The selected clone was sensitive to zeocin (Invitrogen), lacked β-galactosidase activity and showed high tetracycline-inducible, but low basal receptor expression. In these cells, receptor expression was induced by adding 0.5 µg/ml tetracycline in the culture medium lacking the selection antibiotics. Alternatively, receptor expression in mammalian cells was achieved by transient transfections following the protocol described in chapter 4.2.1. The protease inhibitors used (Table 7) were typically added to the culture medium 4h after transient transfections or 1 h after tetracycline induction of stable receptor expression. The cell pellets for protein extraction were collected by harvesting the cells in ice cold phosphate-buffered saline (PBS) and pelleting them by centrifugation at 1,500 g at 4 ˚C. The cell pellets were frozen in liquid nitrogen and stored at -70 ˚C.

Table 7. Protease inhibitors.

Inhibitor Specificity Manufacturer Study Aprotinin Trypsin proteases Fisher Scientific I, II decCMK Proprotein convertases, membrane permeable Tocris Bioscience I, II E-64d Cysteine proteases Sigma-Aldrich I, II GI254023X ADAM10 Tocris Bioscience I, II GM6001 Metalloproteases Enzo Life Sciences I, II GM6001 inactive Metalloproteases Merck Millipore I, II Hexa-D-Arginine Proprotein convertases, membrane impermeable Calbiochem I, II Leupeptin Serine and cysteine proteases Enzo Life Sciences I, II Marimastat Metalloproteases Tocris Bioscience I, II Pepstatin A aspartic proteases Sigma-Aldrich I, II Phosphoramidon Metallo-endopeptidases Sigma-Aldrich I, II Tapi-1 Metalloproteases Merck Millipore I, II Abbreviations: ADAM, a disintegrin and metalloprotease; decCMK, Decanoyl-Arg-Val-Lys-Arg- chloromethylketone.

67 4.2 Cell culture methods

4.2.1 Transient transfections

Transient transfections were performed using linear 25-kDa polyethyleneimine (PEI; Polysciences) (II), Lipofectamine 2000 (I) or 3000 (II) (Thermo Fisher Scientific) or jetOPTIMUS (Polyplus transfection) (II) transfection reagents. For PEI transfection, the transfection reagent and DNA were diluted separately into PBS, mixed by adding PEI/PBS to the DNA/PBS, and the complex formation was allowed to occur for 15 min at 20 ˚C before addition to the cells. The other transfection reagents were used according to the instructions provided by their manufacturers. The cells were transfected with 5 μg (10-cm plate) or 1.5-2 μg (6- cm plate) of DNA using 1:3 or 1:1 DNA-to-reagent ratio. The culture medium was changed 4 h after the transfection and the cells were harvested after 24-48 h.

4.2.2 Small interfering RNA-mediated gene silencing

Gene silencing with small interfering RNA (siRNA) was performed to downregulate ADAM10 and ADAM17 expression in cells (II). Stable HEK293i cells expressing Myc-hGPR37-FLAG were cultured on 10-cm plates for 24 h. The knock-down of ADAM10 and ADAM17 was performed using Lipofectamine RNAi MAX reagent and 20 nM siGENOME SMART pool siRNA against the desired enzymes in Opti-MEM medium containing 4% FBS. The control cells were treated with siGENOME non-targeting siRNA pool. After 24 h, the medium was changed back to DMEM and the cells were cultured for 42 h before 6 h induction of receptor expression.

4.2.3 Cell surface biotinylation

Cell surface biotinylation was used to detect receptors expressed at the plasma membrane by labeling them with the membrane impermeable sulfo-N- hydroxysuccimide (NHS)-biotin reagent (I, II). The labeling enables purification and detection of cell surface proteins using streptavidin-conjugates that bind biotin with high affinity. Before biotinylation, cells were washed twice with PBS containing 10 mM MgCl2 and 100 µM CaCl2 (D-PBS) that prevent cells from detaching from the plate. Cells were then cooled in D-PBS on a metal plate on ice for 10 min and biotinylated for 30 min by adding sulfo-NHS-biotin to the final

68 concentration of 0.5 mg/ml. Excess biotin was quenched by adding Tris-HCl, pH 7.4 to the final concentration of 50 mM and further incubating the cells on ice for 10 min. Finally, the cells were washed with PBS and harvested.

4.2.4 Metabolic pulse-chase labeling

The biosynthesis and maturation kinetics of GPR37 were studied by metabolic pulse-chase labeling experiments (I, II), which enable tracking of the newly synthesized proteins over time. The HEK293i cells were cultured in 25-cm2 cell culture flasks and induced to express hGPR37 for 16 h or as indicated in the results. The cells were depleted in methionine- and cysteine-free DMEM for 1 h at 37 ˚C before pulse-labeling. The newly synthesized proteins were then labeled with 75- 100 µCi/ml [35S]-methionine/cysteine (Easytag Express [35S]-protein labeling mix, PerkinElmer) for 30 min at 37 ˚C. After the pulse, the cells were washed twice with DMEM containing 5 mM methionine to remove the excess label and chased for various time periods at 37 ˚C. Finally, the medium was removed, and the cells were washed and harvested in PBS. When inhibitor-treatments were used in pulse-chase experiments, the drug was added to the depletion, pulse and chase medium.

4.3 Protein extraction and purification

4.3.1 Preparation of whole cell extracts

Total cellular lysates were prepared by suspending the cell pellets in a lysis buffer (Table 8) containing detergent that disrupts the cellular membrane structure. The cells were lysed by mixing at 4 ˚C for 1h and the insoluble material was removed by 30-min centrifugation at 16,000 g (I, II). The protein concentrations were measured using the Bio-Rad DC protein assay reagents according to the instructions provided by the manufacturer. The cell pellets containing [35S]methionine and cysteine-labeled proteins were lysed on a magnetic stirrer at 4 ˚C for 1h followed by centrifugation at 100,000 g (I, II, Fig.5D). No protein concentration measurement was performed with the radioactively labeled samples.

69 Table 8. Main buffer compositions used in the present study.

Buffer Content Membrane preparation buffer 25 mM Tris-HCl, pH 7.4, 20 mM NEM, 2 mM EDTA, 2µg/ml aprotinin, 0.5 mM PMSF, 2 mM 1,10-phenanthroline, 5 µg/ml leupeptin, 5 µg/ml STI, 10 µg/ml benzamidine Membrane preparation buffer B 50 mM Tris-HCl, pH 7.4, 0.5 mM PMSF, 2 mM 1,10- phenantroline, 5 µg/ml leupeptin, 5 µg/ml STI, 10 µg/ml benzamidine Lysis/ membrane solubilization buffer 25 mM Tris-HCl, pH 7.4, 0.5 % DDM/ Triton-X-100, 20 mM NEM, 2 mM EDTA, 140 mM, NaCl, 0.5 mM PMSF, 2 mM 1,10- phenantroline, 5 µg/ml leupeptin, 5 µg/ml STI, 10 µg/ml benzamidine

Endo H deglycosylation buffer 50 mM Na3PO4, pH 5.5, 0.5 % DDM/ Triton-x-100, 50 mM EDTA, 0.2 mM PMSF, 2 mM1,10-phenanthroline, 5 µg/ml leupeptin, 5 µg/ml STI, 10 µg/ml benzamidine

PNGase F deglycosylation buffer 50 mM Na3PO4, pH 5.5, 0.5 % DDM/ Triton-x-100, 50 mM EDTA, 0.2 mM PMSF, 2 mM1,10-phenanthroline, 5 µg/ml leupeptin, 5 µg/ml STI, 10 µg/ml benzamidine Abbreviations: DDM, n-dodecyl-β-D-maltoside; EDTA, ethylenediaminetetraacetic acid; Endo H, Endo-β- N-acetylglucosaminidase H; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; PNGase F, peptide-N-glycosidase F; STI, soybean trypsin inhibitor

4.3.2 Preparation and solubilization of cellular membranes

As GPCRs are membrane-bound proteins, isolated membrane fractions were used as an alternative method for receptor extraction. The membrane fractions do not contain cytosolic proteins, and thus, provide cleaner samples for protein detection. To prepare membranes, the cell pellets were homogenized in membrane preparation buffer (Table 8) using the Ultra-Turrax T-25 homogenizer (IKA) at 19,000 rpm (I, II). Unbroken cells and nuclei were removed from the homogenates by 5 min centrifugation at 1,000 x g and the homogenization and centrifugation steps were repeated. The combined supernatants were centrifuged at 45,000 g for 20 min to obtain the crude membrane fraction and the membranes were washed twice with the homogenization buffer. The membranes were solubilized in membrane solubilization buffer (Table 8) using a 26-G needle and a syringe and by mixing the suspension on a magnetic stirrer at 4 ˚C for 1 h. Finally, the solubilized receptors were separated from the insoluble material by centrifugation at 100,000 g for 1 h. The protein concentrations were determined with the Bio-Rad DC protein assay kit according to the manufacturer’s instructions.

70 The cells carrying [35S]-methionine/cysteine-labeled receptors were homogenized with a tip sonicator in membrane preparation buffer (Table 8) (I). The membrane pellets were obtained by 30 min centrifugation at 40,000 g and washed twice before addition of solubilization buffer (Table 8). The solubilized membranes were subjected to receptor purification directly without protein concentration measurement. Membrane preparation from mouse brain tissue samples was performed using a more simplified protocol (II). The frozen tissue samples were homogenized with the VDI12 homogenizer (VWR) in Membrane preparation buffer B (Table 8) and the homogenates were centrifuged at 12,000 g for 30 min. The membrane pellets were resuspended in the same buffer and the protein concentrations were measured using the Pierce BCA protein assay kit. The proteins were solubilized by adding Laemmli sample buffer (4 % sodium dodecyl sulfate (SDS), 20 % glycerol, 120 mM Tris-HCl, pH 6.8, 0.02 % bromophenol blue) to the membrane suspension in a 1:1 ratio.

4.3.3 Purification of solubilized proteins

The solubilized receptors were purified by one- or two-step immunoprecipitation using antibody agarose resins directed against the N- and C-terminal epitope tags in the receptor constructs (I, II) or ADAM10 (II). The cMyc, HA and FLAG antibody-coupled resins (Sigma-Aldrich) were equilibrated in lysis/membrane solubilization buffer (Table 8) containing 0.1% bovine serum albumin (BSA) at 4 ˚C for 1 h with gentle agitation. Cellular lysates or solubilized membranes were added to the equilibrated resins and incubated in the presence of 0.1% BSA at 4˚C for 2-16 h with rotation. The unbound proteins were removed by washing twice with the lysis/membrane solubilization buffer (Table 8) and four times with the same buffer containing 0.1% detergent. The receptors were eluted from the antibody agarose with 2 x Laemmli sample buffer (Bio-Rad) by incubating the resins first at 22 ˚C for 15 min and then at 95 ˚C for 5 min. The eluates were recovered by 1 min centrifugation at 1,500 g. The receptors subjected to two-step immunoprecipitation were eluted with 1% SDS, 25 mM Tris-HCl, pH 7.4 after the first immunoprecipitation step. The eluates were diluted 10-fold with the lysis/membrane solubilization buffer (Table 8) for the second immunoprecipitation step with fresh antibody resins and finally eluted in 2 x Laemmli sample buffer. In the case of cell surface biotinylation experiments, the biotinylated proteins were first purified with the streptavidin agarose (Thermo

71 Fisher Scientific) and the receptors were then immunoprecipitated with the FLAG antibody. Alternatively, receptor detection after the streptavidin purification was done by western blotting using the FLAG antibody (described in chapter 4.4.1).

4.3.4 Enzymatic deglycosylation

For deglycosylation (I, II), the immunoprecipitated receptors were eluted from the antibody resins with 1% SDS in 50 mM sodium phosphate, pH 5.5 or 7.5. The eluates were diluted 7.5-fold in the appropriate deglycosylation buffer (Table 8) and deglycosylation enzymes endo-β-N-acetylglucosaminidase F (Endo H, Roche Applied Science) and peptide-N-glycosidase F (PNGase F, Roche Applied Science) were added to the maximum concentrations of 50-100 mU/ml and 25-50 U/ml respectively. The enzymatic digestions were performed at 30 ˚C for 16 h and the reactions were stopped by adding 2 x Laemmli sample buffer and cooling the reaction mixtures to 4 ˚C. The membrane fractions prepared from mouse brain tissue were deglycosylated using Endo H and PNGase F reagents from New England Biolabs following the protocol provided by the manufacturer (II).

4.3.5 Purification of the N-terminal receptor fragment from the cell culture medium

The shed N-terminal receptor fragment in the conditioned culture medium was isolated either by immunoprecipitating it from the medium with the cMyc antibody (I, II) or by concentrating the medium by membrane filtration (II). For immunoprecipitation, the stable HEK293i cells were cultured on 10-cm plates and the volume of the culture medium was reduced to 4 ml during the 24-h receptor induction. After induction, the conditioned medium was collected, and cellular debris was removed by 30-min centrifugation at 1,000 g. The collected medium was supplemented with 25 mM Tris-HCl, pH 7.4, 0.5% Triton-X-100, 20 mM N- ethylmaleimide (NEM), 2 mM ethylenediamine tetraacetic acid (EDTA), 2 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM 1,10-phenanthroline, 5 µg/ml leupeptin, 5 µg/ml soybean trypsin inhibitor and 10 µg/ml benzamidine, and a total of 3 ml of each medium sample was subjected to immunoprecipitation with the cMyc antibody resin. The immunoprecipitated receptors were separated using 10-20% Mini-PROTEAN Tris-tricine precast gels (BioRad) and the proteins were detected by western blotting using the cMyc antibody (Table 9).

72 Alternatively, the HEK293 cells were transiently transfected with the Myc- hGPR37-FLAG or hGPR37-NanoLuc for 16 h in serum-free culture medium. The conditioned medium was then collected, centrifuged at 1,000 g and concentrated with Amicon Ultra-15 10K centrifugal filter devices (Millipore). The concentrated medium aliquots were diluted with 2 x Laemmli sample buffer, separated on 12% SDS polyacrylamide gels and analyzed by western blotting using an antibody against the N-terminal domain of hGPR37 (Table 9).

4.4 Protein detection

4.4.1 SDS-PAGE and western blotting

SDS polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins based on their molecular mass (I, II). The protein samples were diluted with Laemmli sample buffer and reduced by heating at 95 ˚C for 2-5 min in the presence of 50 mM dithiothreitol. The reduced proteins were separated on 10-12% SDS-polyacrylamide gels for 60-120 min at 120-150 V using the Mini-PROTEAN 3 electrophoresis cell (Bio-Rad). After SDS-PAGE, the proteins were electroblotted to 0.45 µm polyvinylidene difluoride (PVDF) membranes (Amersham or Immobilon-P, Millipore) using the Mini Trans-Blot cell apparatus (Bio-Rad) at 35 V for 16 h (I, II) or a semidry transfer system (Trans-Blot SD or Trans-Blot Turbo, Bio-Rad) at 20V for 30-60 min (II). After blotting, the molecular weight markers were stained with 0.2% Ponceau, 3% trichloroacetic acid unless prestained markers were used. To prevent nonspecific binding, the membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween (TBS-T) (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) or PBS-T (2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2, 0.05 Tween-20) for 45-60 min at 22 ˚C or overnight at 4 ˚C. The membranes were then probed with the appropriate primary and secondary antibodies (Table 9) to detect the proteins of interest. The antibodies were diluted in TBS/0.1% BSA or PBS-T/5% nonfat dry milk and allowed to bind the proteins for 1 h at 22 ˚C or overnight at 4 ˚C with gentle agitation. The excess antibodies were removed by washing the membranes several times with TBS-T or PBS-T after each antibody incubation. Finally, the proteins were detected with enhanced chemiluminescence and exposed on UltraCruzTM Autoradiography films (Santa Cruz Biotechnology) (I, II) or imaged with the Odyssey Fc imaging system (LI- COR Biosciences) or the Amersham Imager 600 (GE Healthcare) (II).

73 Table 9. Antibodies for western blotting.

Antibody (Species/clonality) Working dilution Blocking Manufacturer Study cMyc (9E10) (Mouse/MC) 1:1000 0.1% BSA Covance I cMyc (9E10) (Mouse/MC) 1:1000 0.1% BSA BioLegend II cMyc (A-14) (Rabbit/PC) 1:1000 0.1% BSA Santa-Cruz I, II FLAG (M2) HRP (Mouse/MC) 1:50000 0.1% BSA Sigma-Aldrich I, II HA (HA-7) HRP (Mouse/MC) 1:5000 0.1% BSA Thermo Fisher Sci. II hGPR37 NT (Rabbit/PC)a 0.5-2 µg/ml 0.1% BSA F. Ciruela II mGPR37 NT (Rabbit/PC)b 0.5-2 µg/ml 0.1% BSA/5%milk F. Ciruela II panGPR37 CT (Rabbit/PC)c 0.5-2 µg/ml 0.1% BSA/5%milk F. Ciruela II ADAM10 (Rabbit/MC) 1:2000 0.1% BSA/5%milk Abcam II ADAM10 (Rabbit/PC) 1:1000 0.1% BSA Abcam II ADAM17 (Rabbit/PC) 1:2000 0.1% BSA Enzo Life Sciences II α-actinin (B-12) (Mouse/MC) 1:1000 5% milk Santa-Cruz II β-actin (Mouse/MC) 1:2000 0.1% BSA Sigma-Aldrich I TRAPA/SSR1 (Rabbit/PC) 1:2000 0.1% BSA Proteintech II Mouse IgG, HRP-linked F(ab’)2 1:40000 0.1% BSA GE Healthcare I, II Rabbit IgG, HRP-linked F(ab’)2 1:40000 0.1% BSA GE Healthcare I, II Goat anti-mouse IgG-HRP 1:10000 5% milk Pierce II Goat anti-rabbit IgG-HRP 1:30000 5% milk Pierce II References: a Gandía et al., 2013 b Lopes et al., 2015 c Morato et al., 2017. Abbreviations: ADAM, a disintegrin and metalloprotease; BSA, bovine serum albumin; CT, C-terminal domain; HRP, Horse radish peroxidase; MC, monoclonal; NT, N-terminal domain; PC, polyclonal; Thermo Fisher Sci., Thermo Fisher Scientific.

The gels containing radiolabeled proteins were stained with Coomassie Brilliant Blue (Bio-Rad) for 1-16 h to visualize molecular weight markers and the excess staining dye was removed with 40% methanol, 10% acetic acid. The gels were then treated with EN3HANCE® liquid autoradiography enhancer (PerkinElmer) for 1 h, washed with water and dried between two sheets of cellophane for 2 h using the Bio-Rad Gel Air Dryer. Protein detection was performed by exposing the gels on Kodak Biomax MR films (Sigma-Aldrich) at -70 ˚C.

4.4.2 Flow cytometry

Flow cytometry provided a method to analyze the expression of full-length receptors with the intact cMyc epitope tag at the surface of live cells (I, II). Depending on the experimental setting, the cells transiently or stably expressing the Myc- and FLAG-tagged hGPR37 were cultured on 6-well-, 24-well- or 10-cm plates. The cells were washed with PBS to remove detached cells and the live cells

74 were collected from the plates and suspended in PBS containing 1% FBS. The receptors at the surface of intact cells were labeled with the cMyc antibody (Table 10) in PBS/1% FBS on ice for 30 min. The negative control cells were treated with PBS/1% FBS in the absence of the primary antibody. To remove the excess antibody, the cells were washed twice with PBS/1% FBS and centrifuged at 200 g for 3 min after each washing step. Next, the cells were stained with the phycoerythrin (PE)-conjugated secondary antibody (Table 10) on ice for 30 min and washed as described above. The dead and broken cells were stained with the 7-amino-actinomycin D (BD Biosciences) and the cells were incubated on ice protected from light for 10 min before analysis on the flow cytometer. Finally, the cMyc-stained cell surface receptors were measured from 10,000 live cells from each sample using the Becton-Dickinson FACSCalibur flow cytometer and the CellQuestTMPro 6.0 software (BD Biosciences) (I, II). Alternatively, the cells were analyzed with the ACCURI C6 flow cytometer (BD Biosciences) and FlowJo V10 software (II).

4.4.3 Confocal microscopy

Confocal microscopy was used to study the cellular localization of GPR37 using fluorescent antibodies (I, II). The cells stably or transiently transfected with hGPR37 were cultured on poly-L-lysine (Sigma) coated glass coverslips on 12- well plates. The cells were washed with PBS and fixed with 100% methanol for 15 min at -20 ˚C. Next, the cells were treated with PBS containing 0.1% Triton-X-100 and 5% BSA for 45 min to permeabilize them for antibodies and to block nonspecific staining. The cells were washed with PBS and the receptors and intracellular marker proteins were probed with the appropriate primary antibodies (Table 10) in PBS/0.1% Triton-X-100/0.1% BSA for 30 min at 22 ˚C. The unbound primary antibodies were removed by PBS washes and the cells were treated with the Alexa Fluor-conjugated secondary antibodies (Table 10) in the dark for 30 min at 22 ˚C. Following the PBS washes, the nuclei were stained with TO-PRO-3 iodine (Invitrogen) for 10 min at 22 ˚C. Finally, the cells were washed with PBS and the coverslips were mounted on glass microscope slides with Immu-mount (Thermo Fisher Scientific). The labeled receptors and marker proteins were detected with the Zeiss LSM780 laser scanning confocal microscope (Carl Zeiss Microscopy) using the Plan-Apochromat 63x1.4 numerical aperture oil-immersion objective.

75 Table 10. Antibodies for flow cytometry and confocal microscopy.

Antibody (Species/clonality) Working dilution Application Manufacturer Study cMyc (9E10) (Mouse/MC) 1:300 FC, IF Covance I, II cMyc (9E10) (Mouse/MC) 1:500 FC BioLegend II cMyc (Rabbit/PC) 1:250 IF Sigma-Aldrich I FLAG (M2) (Mouse/MC) 1:10000 IF Sigma-Aldrich I Calreticulin (Rabbit/PC) 1:1000 IF Stressgen I ERGIC53 (Mouse/MC) 1:500 IF Enzo Life Sciences I GM130 (Rabbit/PC) 1:500 IF BD Biosciences I Na/K ATPase (Mouse/MC) 1:5000 IF Abcam I, II Sec61β (Rabbit/MC) 1:500 IF Merck Millipore I TGN46 (Sheep/PC) 1:200 IF AbD Serotec I Alexa Fluor 488 goat anti-mouse 1:250 IF Invitrogen I Alexa Fluor 488 goat anti-rabbit 1:250 IF Invitrogen I, II Alexa Fluor 568 goat anti-mouse 1:250 IF Invitrogen I, II Alexa Fluor 568 goat anti-rabbit 1:250 IF Invitrogen I Alexa Fluor 568 donkey anti-sheep 1:250 IF Invitrogen I PE-conjugated rat anti-mouse IgG1 1:100 FC BD Biosciences I, II Abbreviations: FC, flow cytometry; IF, immunofluorescence (confocal) microscopy; MC, monoclonal; PC, polyclonal; PE, phycoerythrin.

4.4.4 N-terminal sequencing

The N-terminal sequencing of the cleaved receptors was performed to identify the metalloprotease cleavage site at the GPR37 ectodomain (I). Cellular membranes were prepared from stable HEK293i cells that were induced to express the Myc- and FLAG-tagged hGPR37 for 24 h. The receptors were purified by two-step immunoprecipitation with the FLAG antibody-resin and eluted with 200 µg/ml of FLAG peptide (Sigma-Aldrich). The eluate was concentrated in three 300 µl aliquots using Amicon Ultra 30 K centrifugal filter devices. The receptors were then separated on 10% SDS-PAGE gels and electroblotted on ProBlot membranes (Thermo Fisher Scientific). The proteins were visualized by Serva Blue staining solution (0.1% Serva Blue, 1% acetic acid, 40% ethanol) and the protein bands representing the cleaved membrane-bound receptor fragment were excised from the membrane and subjected to sequence analysis. The N-terminal sequence was determined by five cycles of automated Edman degradation on the protein sequencer ProciseTM 492 (Thermo Fisher Scientific).

76 4.5 Data analysis

The sequence alignment of the N-terminal domains of hGPR37 and mGPR37 (II, Fig. S4) was performed using the Clustal Omega 1.2.4 multiple sequence alignment program. The sequence logo of the GPR37 N-terminus (II, Fig. 6) was generated with WebLogo (Crooks, Hon, Chandonia, & Brenner, 2004). Clustal Omega multiple sequence alignment was performed for the respective sequences of fourteen placental mammals (II, Table S1) and the alignment was submitted to WebLogo for the generation of a graphic presentation. The western blot and fluorography films were analyzed by densitometric scanning using the Umax PowerLook 1120 color scanner (Amersham Biosciences) and ImageMaster 2D Platinum 6.0 software (I, II). Alternatively, the chemiluminescence of western blots was detected by digital imaging with the LI- COR Odyssey Fc imager or the Amersham Imager 600 (II). Relative intensities of the desired protein bands were quantified with the Image J 1.45 or Image Studio v. 5.2 software. Local background was subtracted from the quantified uncalibrated optical densities. The relative molecular masses of the receptor species were calculated with the GraphPad Prism v. 4.01-v.7.03 software by comparing their electrophoretic mobilities to those of the known standard proteins (I, II). The data were analyzed with the GraphPad Prism v. 4.01-v.7.03 software (I, II). The calculations were performed by subtracting the background fluorescence of live cells stained only with the secondary antibody from the mean fluorescence of live cells stained with the primary and secondary antibodies. Statistical analyses were carried out using one-way analysis of variance followed by Bonferroni’s, Dunnett’s or Tukey’s post hoc multiple comparison test. The limit for statistical significance was set at p < 0.05.

77

78 5 Results

5.1 Biosynthesis and expression of GPR37 in mammalian cells

GPR37 was originally associated with early onset Parkinson’s disease when it was identified as a substrate of the parkin ubiquitin ligase and reported to aggregate in cells causing cell toxicity (Imai et al. 2001). This, together with the low levels of GPR37 typically detected at the cell surface, led researchers to hypothesize that the receptor folds inefficiently and is therefore retained in the ER. Consequently, several efforts have been made to increase the plasma membrane trafficking of GPR37 and to reduce receptor-induced cell toxicity. However, the biosynthetic profile of GPR37 and the mechanisms that regulate the receptor have remained poorly characterized. In this thesis, the steps that govern GPR37 maturation, expression and processing were studied using mammalian cell models and various cell biological and biochemical methods.

5.1.1 Characterization of the expressed GPR37 species (I)

To characterize the expressed GPR37 species under steady-state conditions, the hGPR37 was modified to contain an HA signal peptide, an N-terminal Myc epitope and a C-terminal FLAG epitope (I, Fig. 1A). The construct was expressed in stably transfected tetracycline-inducible HEK293i cells to enable receptor expression in a controlled manner. The expressed receptor forms were analyzed by western blotting after immunoprecipitation with the FLAG antibody. The cMyc antibody revealed two specific bands of about 67 kDa and 96 kDa, as well as higher molecular weight bands most likely representing receptor oligomers (I, Fig. 1B and D). As in the case of the cMyc antibody, the 67-kDa and 96-kDa species were detected with the FLAG antibody, although the intensity of the 96-kDa receptor form was typically weak and hard to detect (I, Fig. 1B and D). Additionally, the FLAG antibody revealed a very intense and heterogeneous band of 53 kDa and a less abundant species of 34 kDa. Unlike the 67-kDa and 96-kDa species, these receptor forms became more apparent with increasing induction time (I, Fig. 1D). As they were not recognized with the cMyc antibody, they were identified as proteolytically processed forms of GPR37 missing part of the extracellular N-terminal domain that contains the Myc epitope.

79 To characterize the expressed receptor forms more specifically, enzymatic deglycosylation with PNGase F and Endo H were performed. This approach takes advantage of the unique specificities of the two deglycosylation enzymes and was used to investigate the maturation state of the receptor species. While Endo H is able to cleave only glycans typical for ER-localized glycoproteins, PNGase F can release unprocessed glycans in ER-localized proteins as well as N-glycans that have been additionally modified in the Golgi compartment (Petäjä-Repo et al., 2000). The 67-kDa GPR37 species was sensitive to both Endo H and PNGase F, indicating that it represents an ER-localized receptor precursor (I, Fig. 1C). In contrast, the 53-kDa and the 96-kDa species were deglycosylated with PNGase F only (I, Fig. 1C). Therefore, they were identified as mature receptor forms carrying N-glycans that have been further processed in the Golgi. In summary, we identified two full- length (precursor and mature) and two N-terminally processed GPR37 species. These species and additional receptor forms described later in this thesis (see 5.2.3 and 5.3.2) are summarized in Table 11 (see also II, Fig. 2B).

Table 11. Identification of the expressed hGPR37 species in HEK293 cells.

MW (kDa) Identification Antibody detection Study 120-200 Oligomeric species cMyc, FLAG, NT Ab, CT Ab I, II 96 Mature receptor, full-length cMyc, FLAG, NT Ab, CT Ab I, II 70 Mature receptor, cleaved at Arg54↓Arg55 FLAG, NT Ab, CT Ab II 67 Receptor precursor cMyc, FLAG, NT Ab, CT Ab I, II 53 Mature receptor, cleaved at Glu167↓Gln168 FLAG, CT Ab I, II 32-35 Shed N-terminal fragment cMyc, NT Ab I, II 34 Mature receptor, cleaved close to TM1 FLAG, CT Ab I, II Abbreviations: CT/ NT Ab, C-terminal/ N-terminal GPR37 antibody; kDa, kilodalton; MW, molecular weight; TM, transmembrane domain.

5.1.2 Maturation of GPR37 (I, II)

The metabolic pulse-chase labeling using HEK293i cells stably expressing the receptor revealed that GPR37 displays fast maturation kinetics (I, Fig. 2A-B and II, Fig. 5A, upper panel). The 67-kDa receptor precursor was clearly detectable at the end of the pulse, but disappeared completely within 4 h (I, Fig. 2A-B and II, Fig. 5A). The mature 96-kDa species became visible after the 30-min chase. However, it started to disappear already after 1 h, while the cleaved 53-kDa species and the less abundant 34-kDa species became apparent (I, Fig. 2A and II, Fig. 5A). No changes in the relative amount of the cleaved receptor species were detected when

80 the samples were analyzed under non-reducing conditions (I, Fig. 2C). This finding indicates that the N-terminal fragment is not held together with the membrane- bound receptor fragment by disulfide-bonding between Cys-residues as has been shown for some other GPCRs that undergo N-terminal cleavage (Rapoport & McLachlan, 2016). These data together imply that GPR37 is cleaved efficiently shortly after receptor maturation into a species containing fully modified N-glycans.

5.1.3 Characterization of transiently expressed receptors (I, II)

To test if the observed receptor cleavage depends on the expression system used or varies between human and mouse receptors, transient transfections of hGPR37 and mGPR37 into HEK293 and SH-SY5Y human neuroblastoma cells were performed. Western blot analysis revealed that hGPR37 is cleaved in these cell lines (I, Fig. 3A-B and II, Fig. 2E-F), and similar results were obtained using MEF and LoVo cells (II, Fig. 4A and 7F) Moreover, the mGPR37 was also cleaved in transiently transfected HEK293 cells (I, Fig. S2 and II, 2F and S1). However, the size of the cleaved receptor species was larger in mGPR37 than that observed with hGPR37 (II, Fig. 2F and S1), indicating that there are some differences in receptor cleavage between the species. Interestingly, transient GPR37 expression led to an increased accumulation of higher molecular mass receptor species at the top of the gel (I, compare Figs. 3A and 1B). These results are consistent with previously published findings (Imai et al., 2001; Rezgaoui et al., 2006), and indicate that GPR37 shows an increased tendency to aggregate in transient expression systems compared to the stable receptor expression. Despite these differences between the stable and transient expression systems, these results imply that the receptor cleavage is not a cell type or species-specific event.

5.2 Processing of GPR37 at the major cleavage site

Our results on GPR37 biosynthesis revealed that the receptor is subject to N- terminal cleavage rapidly after its maturation. As a result, the majority of receptors detected under steady-state conditions are in the N-terminally truncated form. This finding provides an explanation for the previously reported poor receptor expression at the cell surface as several studies have relied on receptor detection using N-terminal antibodies or N-terminally placed epitope tags. To better understand the mechanism of GPR37 cleavage, several experiments were

81 performed to characterize the enzymes, cleavage sites and cellular localization of the full-length and cleaved receptor species.

5.2.1 Cellular localization of GPR37 species (I, II)

The cellular localization of full-length and N-terminally processed GPR37 species was studied by confocal microscopy. The cMyc-stained full-length receptors localized mainly in intracellular compartments (I, Fig. 4A-D and II, Fig. 1B, top panel), while the receptor species stained with the FLAG antibody where mostly found at the cell surface (I, Fig. 4A-B). Since the FLAG antibody detects predominantly the cleaved receptor species (I, Fig. 1B), most of the receptors at the cell surface are likely to represent N-terminally processed receptor forms. This conclusion was further supported by cell surface biotinylation assays, which showed that the cleaved form was the predominant receptor species at the cell surface (I, Fig. S3 and II, Fig. 5A). The subcellular localization of the full-length receptors was studied more specifically using marker proteins for various cellular compartments. Some degree of co-localization was observed between the receptor and the marker proteins for ER, ERGIC and cis-Golgi (I, Fig. 5A-D). However, the full-length receptors appeared to localize mainly in the trans-Golgi compartment (I, Fig. 5E). These results together imply that GPR37 remains in its full-length form in the secretory pathway from the ER to the TGN and is cleaved only after it leaves the latter compartment.

5.2.2 Metalloprotease cleavage of GPR37 (I, II)

The enzyme family responsible for GPR37 cleavage was identified using protease inhibitors designed to reduce the activity of specific enzyme groups. Metalloprotease inhibitors GM6001 and marimastat significantly increased the abundance of full-length mature receptors detected by western blotting, but inhibitors against cysteine, serine or aspartyl proteases had no substantial effect (I, Fig.6A). Correspondingly, the amount of the cleaved 53 kDa species was decreased after metalloprotease inhibitor treatment (II, Fig.2G). However, the abundance of full-length receptors was also increased by the furin inhibitor decanoyl-RVKR- chloromethylketone (decCMK), which is able to inhibit furin and other proprotein convertases (I, Fig.6A). Further evidence for these findings was obtained by flow cytometry and confocal microscopy. Active metalloprotease inhibitors and decCMK increased the amount of cMyc-stained full-length receptors at the cell

82 surface, while inactive GM6001 or a membrane impermeable proprotein convertase inhibitor had no effect (I, Fig. 6B-D and II, Fig. 1B-C). These results together imply that GPR37 processing is mediated by an enzyme that belongs to the metalloprotease family. However, furin or some other proprotein convertase might have an indirect effect on GPR37 cleavage as several metalloproteases are known to require prodomain removal by proprotein convertases to gain enzymatic activity (Weber & Saftig, 2012).

5.2.3 Ectodomain shedding and cleavage site identification (I)

To test if the cleaved GPR37 ectodomain is shed from the cells, conditioned medium from HEK293i cells expressing GPR37 was subjected to immunoprecipitation and the purified species were analyzed by western blotting with the cMyc-antibody. The antibody recognized a specific band of about 32 kDa from the medium when the cells were induced to express GPR37 (I, Fig. 7A). However, this species was not detected if the metalloprotease activity was inhibited by marimastat. Therefore, it was concluded that GPR37 cleavage is followed by ectodomain shedding (see Table 11 and II Fig. 2B for the identification of the 32- kDa species). To rule out the possibility that the observed receptor cleavage and ectodomain shedding were affected by the added epitope tags in the receptor constructs, two control constructs were created. One had an intact N-terminus without the Myc-epitope and contained the endogenous signal peptide, and the other one was missing the C-terminal FLAG-epitope (I, Fig. 7B). As anticipated, the receptor cleavage and ectodomain shedding were found to occur normally despite these modifications (I, Fig. 7C-E). Given that the GPR37 N-terminus contains three putative N-glycosylation sites at Asn36, Asn222 and Asn239, the location of the metalloprotease cleavage site was first estimated in relation to these sites. Toward this end, the 53-kDa C-terminal cleaved fragment and the 32-kDa shed N-terminal fragment were subjected to de- N-glycosylation with PNGase F. Sequential deglycosylation of the 53-kDa species revealed that it carries two N-glycans and the 32-kDa species was also found to be N-glycosylated (I, Fig. 8A-B). These results indicate that all three N-glycosylation sites in the GPR37 ectodomain are used and the cleavage site is located somewhere between Asn36 and Asn222. Finally, the cleavage site location was identified more specifically between Glu167 and Gln168 by N-terminal sequencing of the cleaved 53-kDa receptor fragment (I, Fig. 8C). However, point mutations and short deletions of the amino acids surrounding the cleavage site were not sufficient to

83 prevent receptor cleavage (I, Fig. 8D). Similar results were obtained upon larger deletions around the identified cleavage site (Fig. 10). This finding is not surprising, as substrate selectivity of metalloproteases is often defined by protein conformation and distance of the cleavage site from the cell membrane rather than a specific cleavage site recognition motif (Matthews et al., 2017; Riethmueller et al., 2016).

Fig. 10. Deletions at the identified Glu167↓Gln168 cleavage site do not prevent GPR37 cleavage. The wild-type (WT) GPR37 and the indicated mutant receptor constructs were transiently transfected into HEK293S cells for 24 h. Receptors were immunoprecipitated (IP) from cellular lysates and analyzed by western blotting (WB) using the FLAG antibody. Symbols: closed circle, receptor precursor; open square, cleaved 53 kDa species.

5.3 Identification of the enzymes responsible for GPR37 cleavage

The experiments with broad-range protease inhibitors indicated that GPR37 is cleaved by a metalloprotease. To characterize the cleaving enzyme more specifically, assays using more selective metalloprotease inhibitors and downregulation and over-expression methods were employed. Metalloproteases ADAM10 and ADAM17 are commonly implicated in ectodomain shedding. ADAM10, in particular, is known to cleave several substrate proteins in a constitutive manner without external stimulation (Saftig & Lichtenthaler, 2015). Therefore, ADAM10 was selected as an initial candidate to test in our experimental settings.

84 5.3.1 ADAM10 cleavage between Glu167 and Gln168 (II)

The identification of the cleaving enzyme was initiated using an inhibitor-based approach. Toward this end, the ability of a broad-spectrum metalloprotease inhibitor marimastat and an ADAM10-selective inhibitor GI254023X to reduce receptor cleavage was tested. The Myc-and FLAG-tagged GPR37 was expressed in stable HEK293i cells. Confocal microscopy analysis of the GPR37 localization revealed that inhibitor treatment increased the number of the cMyc-stained full- length receptors at the plasma membrane (II, Fig. 1B). This was observed as increased colocalization with the marker protein for plasma membrane. A similar increase in the amount of full-length receptors at the plasma membrane was detected by flow cytometry when the ADAM10-selective inhibitor was used (II, Fig. 1C). The effect was also dependent on the inhibitor concentration used. The effect of metalloprotease-inhibitors on GPR37 cleavage was characterized more precisely by western blotting using antibodies directed against the N- and C- terminal epitope tags or the receptor N-terminal domain. Analysis of the expressed receptor species revealed that GI254023X reduced the amount of the shed 35-kDa receptor fragment (II, Fig. 2A) and the membrane-bound 53-kDa cleaved receptor (II, Fig. 2C-D) species in a concentration-dependent manner. Correspondingly, the abundance of the 96-kDa full-length receptor was increased (II, Fig. 2C-D). Similar results were also obtained when the Myc- and FLAG-tagged hGPR37 or C- terminally FLAG-tagged mGPR37 were expressed transiently in inhibitor-treated SH-SY5Y and HEK293 cells (II, Fig. 2E-F). Although metalloprotease inhibitors had a clear effect on the 53-kDa receptor species, the inhibitors did not reduce the amount of the smaller 34-kDa membrane- bound cleaved species (II, Fig. 2C). In contrast, this receptor form became more abundant when the cells were treated with cysteine protease inhibitors leupeptin and E-64d (II, Fig. 2G and 5C). Therefore, it was considered to represent a degradation intermediate in the endo-lysosomal pathway where cysteine proteases are active. This hypothesis, however, was not investigated further. Another interesting finding was that an additional species of about 70 kDa was detected with the FLAG antibody when the cells were treated with the metalloprotease inhibitors (II, Fig. 2C and G). That observation indicates that the N-terminus of GPR37 contains an additional cleavage site upstream from the Glu167↓Gln168 metalloprotease site. Since the results with GI254023X indicated that ADAM10 is involved in GPR37 processing at the Glu167↓Gln168 cleavage site, this possibility was further

85 investigated by knock-down and knock-out experiments. Western blotting and flow cytometric studies revealed that siRNA-mediated downregulation of ADAM10, but not ADAM17, reduced the cleavage and shedding of the GPR37 ectodomain in

HEK293i cells stably expressing GPR37 (II, Fig. 3A-D). Correspondingly, transient expression of GPR37 in HEK293 and MEF cells lacking ADAM10 or ADAM17 showed that the receptor cleavage was significantly reduced in cells that did not express ADAM10 (II, Fig. 4A-B). Furthermore, overexpression of HA-tagged ADAM10 in ADAM10-/- HEK293 cells was able to rescue the enzymatic activity and restore the cleavage of GPR37 at the Glu167↓Gln168 site (II, Fig. 4D). As expected, overexpression of the inactive ADAM10 Glu384Ala mutant had no effect. Finally, the cleavage of mGPR37 was reduced in ADAM10-/- HEK293 cells as was the case with hGPR37 (II, Fig. 4E). In summary, these findings imply that ADAM10 is indeed the major enzyme responsible for GPR37 cleavage.

5.3.2 Furin cleavage between Arg54 and Asp55 (II)

The western blot results obtained in the presence of metalloprotease inhibitors suggested that the ectodomain of hGPR37 is processed at an additional site to the previously identified Glu167↓Gln168 cleavage site. A receptor species of about 70 kDa was detected with the FLAG and GPR37 N-terminal antibodies when the cells were treated with metalloprotease inhibitors GI254023X, GM6001, TAPI-1 or marimastat, but not upon treatment with other types of protease inhibitors (II, Fig. 2C, G-H). The 70-kDa species, however, was not recognized by the cMyc antibody that only detects the Myc-epitope at the N-terminal end of the receptor (II, Fig. 2H). Based on these observations, the ectodomain of hGPR37 was hypothesized to be cleaved by a non-metalloprotease enzyme at a site located upstream from the identified metalloprotease cleavage site (see Table 11 and II, Fig. 2B). The identity of the 70-kDa receptor species was characterized by metabolic pulse-chase labeling experiments. To gain a better understanding about the maturation kinetics and cell surface appearance of this species, the HEK293i cells stably expressing hGPR37 were pulse-labeled and chased for various periods of time in the presence or absence of GI254023X and the cell-surface receptors were biotinylated at the end of the chase. Under control conditions, the receptor showed normal, rapid, maturation kinetics and was processed into the 53-kDa cleaved form and the less abundant 34-kDa species (II, Fig.5A). However, the 70-kDa species became apparent when the cells were treated with GI254023X. Furthermore, streptavidin purification of the biotinylated receptors revealed that this species was

86 present at the cell surface together with the 96-kDa full-length and 53-kDa cleaved receptor forms. The 70-kDa receptor form was insensitive to deglycosylation with Endo H but sensitive to PNGase F (II, Fig. 5B), indicating that it represents a mature that has been proteolytically processed close to the N- terminal end. The protease family responsible for the conversion of hGPR37 to the 70-kDa species was characterized by screening the inhibitory effect of various protease inhibitors on this cleavage step. Treatment with the membrane permeable proprotein convertase inhibitor decCMK prevented receptor cleavage, while no effect was observed with the membrane impermeable proprotein convertase inhibitor or inhibitors against other protease families (II, Fig. 5C-D). These data imply that GPR37 is processed by a proprotein convertase enzyme and the cleavage takes place before the receptor reaches the cell surface. Inhibition of the proprotein convertase activity using decCMK was additionally found to increase the abundance of the shed receptor fragment (II, Fig. 7D-E). This finding, together with the observation that the 35-kDa fragment could be recognized by the cMyc antibody that detects the N-terminal Myc-tag (I, Fig. 7A, D and II, Fig. 2A and 3B), indicates that the fragment originates from receptors that are cleaved by ADAM10 but not by proprotein convertases. Finally, the 70-kDa species was not detected when hGPR37 was transiently transfected into furin-deficient LoVo cells (II, Fig. 7F), indicating that furin is the major proprotein convertase responsible for GPR37 processing. The location of the proprotein convertase cleavage site in the GPR37 ectodomain was identified based on sequence analysis and site-directed mutagenesis. The sequence of the hGPR37 N-terminus contains several potential sites with the common proprotein convertase recognition motif (Arg/Lys)-2nx- Arg/Lys↓ (where n = 0–3 amino acids). Four of them are located upstream of the Glu167↓Gln168 metalloprotease cleavage site and they are well conserved among mammalian species (II, Fig. 6). Deletion and point mutations at the most N- terminally located site (Arg51-54) abolished the proprotein convertase cleavage of GPR37 (II, Fig. 7A-C). Therefore, the Arg54↓Asp55 site was identified as the proprotein convertase cleavage site in the receptor ectodomain. The identified cleavage sites in the hGPR37 ectodomain and the cleaving enzymes are illustrated in Figure 11.

87 Fig. 11. N-terminal cleavage of hGPR37. The three cleavage sites in the hGPR37 ectodomain are marked with scissors. Furin and ADAM10 were identified as the main enzymes that cleave GPR37 at the Arg54↓Asp55 and Glu167↓Gln168 sites, respectively. GPR37 is additionally cleaved at an unidentified site located downstream from the N- glycosylation sites Asn222 and Asn239.

5.4 ADAM10 as a physiologically relevant GPR37 cleaving enzyme (II)

Given that the ADAM10-mediated cleavage was found to affect both human and mouse GPR37 in cultured cells, the receptor processing was studied in a more physiologically relevant environment. Toward this end, tissue samples were collected from various brain regions of GPR37 knock-out and corresponding wild- type mice and the expressed GPR37 species were analyzed by western blotting. The N-terminal antibody recognized a specific band of about 62 kDa from the striatum, hippocampus and cerebellum of GPR37 wild-type but not from GPR37 knock-out mice (II, Fig. 8). In contrast, a smaller species of about 42 kDa was detected with the C-terminal antibody. These results imply that GPR37 is subject to N-terminal processing also in native brain tissue. Finally, to study the role of ADAM10 in the cleavage of GPR37 in the mouse brain, tissue samples from cortex, hippocampus and cerebellum of conditional ADAM10 knock-out mice (ADAM10 cKO) were analyzed by western blotting.

88 Since ADAM10-deficient mice die already at embryonic day 9.5 (D. Hartmann et al., 2002), ADAM10 deletion in these mice was targeted to the postnatal neurons in specific brain regions (Prox et al., 2013). In line with previously published findings (Prox et al., 2013), the expression of both mature and immature forms of ADAM10 was significantly decreased in the targeted brain regions, cortex and hippocampus, compared to the littermate controls (II, Fig. 9 B and E). In contrast, no obvious difference was observed in the cerebellum that served as a nontargeted control region (II, Fig. S3 B). Analysis of the expressed GPR37 species with the N- terminal antibody revealed that the abundance of the 62-kDa species was considerably increased in the targeted regions (II, Fig. 9 A, C-D and F). No such increase, however, was observed in the cerebellum (II, Fig.S3 A and C). Therefore, ADAM10 appears to play an important role in the processing of GPR37 also in the mouse brain.

89

90 6 Discussion

6.1 N-terminal cleavage of GPR37

The previous studies suggesting that GPR37 may be prone to inefficient folding and intracellular aggregation (Imai et al., 2001; Kitao et al., 2007) prompted us to investigate the biosynthesis and molecular mechanisms that could regulate the receptor in a more detailed manner. Western blot characterization of the expressed GPR37 species in stably and transiently transfected HEK293 cells led to the unexpected discovery that the receptor is subject to proteolytic processing in its large N-terminal domain. Using site-directed mutagenesis and N-terminal sequencing, two N-terminal cleavage sites were identified at Arg54↓Asp55 and Glu167↓Gln168. Western blotting revealed that GPR37 is additionally processed at a third site most probably located close to the first TM domain. These findings were confirmed by various methodological approaches that also demonstrate that receptor processing takes place in vivo. In western blots, increased induction time of receptor expression led to increased accumulation of the cleaved receptor species. Cleaved GPR37 species were also observed in pulse-chase experiments shortly after receptor maturation. Moreover, an N-terminal receptor fragment could be recovered from the conditioned cell culture medium, and inhibition of GPR37 cleavage resulted in reduced ectodomain shedding. Confocal microscopy, flow cytometry and cell surface biotinylation assays further demonstrated that the majority of receptors at the plasma membrane were cleaved at the Glu167↓Gln168 site, whereas inhibition of receptor cleavage led to increased levels of full-length GPR37 detected at the cell surface. Importantly, GPR37 was cleaved at the Glu167↓Gln168 site in several cell lines in addition to HEK293 cells and receptor cleavage was also observed in mouse brain tissue. Thus, N-terminal cleavage of GPR37 appears to be a physiologically relevant event that might contribute to the activation mechanism or functional regulation of the receptor. Metabolic pulse-chase labeling analysis revealed that GPR37 displays rapid maturation kinetics in stably transfected HEK293 cells, and the receptor is quickly converted into the cleaved species. Although all three cleaved receptor forms could be detected, the receptor form cleaved at the Glu167↓Gln168 site was the predominant species. These observations allow a conclusion that GPR37 is exported from the ER and matures efficiently in a stable expression system. This finding was somewhat surprising as previous reports have suggested that unfolded

91 GPR37 tends to aggregate intracellularly and the receptor is poorly expressed at the cell surface (Dunham et al., 2009; Imai et al., 2001). Since the previous studies have typically relied on transient overexpression of the receptor, the observed differences can be partially explained by dissimilar expression systems. In line with this notion, we observed accumulation of higher molecular mass aggregates at the top of SDS-PAGE gels upon transient receptor expression. Thus, GPR37 might be comparatively sensitive to disturbances under cellular stress conditions, which could become more apparent upon transient receptor expression. Likewise, cellular changes in Parkinson’s disease and other neurological disorders might cause GPR37 to fold inefficiently and make the receptor more aggregation prone. On the other hand, our results do not support the hypothesis that the low levels of GPR37 detected at the plasma membrane result from impaired folding and maturation under stable conditions. Instead, it is more likely that the cleaved receptor form, that is the most prevalent receptor species at the cell surface, has not been detected in previous studies because N-terminally added epitope tags or N-terminally targeted antibodies were commonly used for receptor detection. While the possibility that GPR37 might be subject to N-terminal processing has not been considered before, several previous studies support this idea. First, a receptor species with a molecular size similar to the N-terminally cleaved 53-kDa receptor form has been detected in natural tissues as well as in diverse heterologous expression systems using antibodies detecting the C-terminal domain of GPR37 (Imai et al., 2001; Omura, Kaneko, Tabei, Okuma, & Nomura, 2008; Rezgaoui et al., 2006). Secondly, the expression pattern of mGPR37 published previously (Imai et al., 2001) is very similar to that reported in the present work. Interestingly, in one of the studies aiming to increase cell surface trafficking of hGPR37, Dunham and her colleagues demonstrated that the plasma membrane expression of GPR37 could be enhanced significantly by 210 or 255 amino acid-long N-terminal truncations, but not with shorter truncations (Dunham et al., 2009). As the study relied on receptor detection using an N-terminally placed FLAG epitope, it is likely that the naturally occurring receptor cleavage led to the loss of epitope tag when it was placed N-terminally to the cleavage site. Finally, our finding that GPR37 undergoes ectodomain shedding is supported by studies in which peptides corresponding to the N-terminal region of GPR37 were identified in the human cerebrospinal fluid (Noben et al., 2006; Shi et al., 2015). Additionally, an O-glycosylated peptide corresponding to the GPR37 N-terminal domain has been found in the secretome of HEK293 cells (Steentoft et al., 2013).

92 As mentioned in chapter 2.3.3, a small number of class A GPCRs have been demonstrated to undergo N-terminal proteolytic processing. Intriguingly,

GPR37L1 and ETBR, two receptors with the highest sequence similarity to GPR37, are among these N-terminally cleaved receptors (Coleman et al., 2020; Grantcharova et al., 2002; Kozuka, Ito, Hirose, Lodhi, & Hagiwara, 1991). Several peptides corresponding to the N-terminal domain of GPR37L1 have also been discovered in the human cerebrospinal fluid (Halim, Ruetschi, Larson, & Nilsson, 2013; Noben et al., 2006), indicating that cleaved receptor fragments can be released from cells as are the GPR37 cleavage products. Thus, N-terminal cleavage appears to be a common mechanism to regulate these three highly homologous GPCRs and might also be involved in the regulation of other receptors related to

GPR37. However, the N-terminal domains of GPR37, GPR37L1 and ETBR are very different. While GPR37 possesses an unusually long N-terminal tail, the N- termini of GPR37L1 and ETBR are much shorter (Sakurai et al., 1990; Valdenaire et al., 1998). Additionally, the expression pattern in different tissue and cell types varies between the receptors (N. J. Smith, 2015). Therefore, the mechanism and functional role of the cleavage may also differ considerably. Additionally, the results of the present work imply that there might be some species-specific differences in the processing of GPR37 considering that the cleaved human receptor was smaller in size than the mouse receptor. Therefore, it is possible that the cleavage sites are not identical in different species or the receptors are differentially regulated. Alternatively, the GPR37 ectodomain might be prone to a series of proteolytic cleavage events in addition to those identified in the present study. Consequently, the precise pattern of these cleavage steps may vary between species or even within the same species in different cell types or under dissimilar cellular conditions.

6.2 Enzymes responsible for GPR37 cleavage

We identified ADAM10 and furin as the key enzymes cleaving GPR37 at the Glu167↓Gln168 and the Arg54↓Asp55 sites, respectively. This finding was quite extraordinary as no previous reports exist validating ADAM10 as a cleaving enzyme for multi-spanning membrane proteins, such as GPCRs. Although some GPCRs are reported to be cleaved by metalloproteases, these studies typically lack characterization of the precise enzymes involved in the process. The cleavage and shedding of the TSHR ectodomain have been demonstrated in several studies, but no consensus exists about the metalloprotease responsible (Rapoport & McLachlan,

93 2016). The finding that the TSHR ectodomain contains a peptide sequence similar to known ADAM10 substrates led researchers to suggest that ADAM10 is the TSHR cleaving enzyme (Kaczur et al., 2007). However, this hypothesis has not been experimentally corroborated. Additionally, ADAM17 or a related protease has been suggested as the enzyme responsible for PAR1 ectodomain shedding (Ludeman, Zheng, Ishii, & Coughlin, 2004), but again without direct experimental evidence. More recently, various MMPs have been reported to cleave and activate PAR1 (Hamilton & Trejo, 2017; Heuberger & Schuepbach, 2019). Additionally, MMP14 can cleave adhesion GPCR B1 (Cork et al., 2012). Metalloprotease- mediated cleavage has also been demonstrated for ETBR and GPR37L1 that are closely related to GPR37 (Coleman et al., 2020; Grantcharova et al., 2002). However, the precise enzymes are not known. Moreover, metalloproteases are also implicated in the cleavage of β1-adrenergic receptor (Hakalahti et al., 2010), V2 vasopressin receptor (Kojro & Fahrenholz, 1995), parathyroid hormone receptor (Klenk et al., 2010), sphingosine 1-phosphate receptor 2 (El Buri et al., 2018) and C5a receptor (van den Berg et al., 2012), all of which belong to the Class A/ rhodopsin GPCR family. In the present study, the ADAM10-selective inhibitor GI254023X and ADAM10 deficient HEK293 and MEF cells were used to demonstrate that ADAM10 cleaves GPR37 at the Glu167↓Gln168 site. Furthermore, over- expression of ADAM10 rescued GPR37 cleavage in ADAM10 knock-out cells. Importantly, conditional depletion of ADAM10 in neuronal cells of mouse cortex and hippocampus reduced the cleavage of endogenous GPR37. Although the role of ADAM10 in the proteolytic processing of GPR37 was demonstrated by various experimental settings, this study did not investigate the possible involvement of other metalloproteases in the cleavage of GPR37. Thus, it is possible that ADAM10 is not the only metalloprotease that can cleave GPR37 at the identified site or some other N-terminal site. For instance, other ADAM family members may compensate for ADAM10 activity under certain conditions. This may occur, for example, during cellular stress or if ADAM10 expression is compromised. Other proteases may also cleave GPR37 in an inducible manner, whereas ADAM10-mediated cleavage occurs constitutively. The present work leaves some open questions about the cleavage mechanism of GPR37 by ADAM10. ADAM10 is a membrane-tethered protease and the cleavage site in the substrate protein is typically located in close proximity to the cellular membrane. However, the identified Glu167↓Gln168 cleavage site in the GPR37 ectodomain is positioned almost 100 amino acids apart from the first TM

94 region. The long distance between the cleavage site and the first TM domain raises a question about cleavage site accessibility to ADAM10. Obviously, GPR37 is an unusual ADAM10 substrate as no previous analyses of GPCRs cleaved by ADAM proteases exist to provide a reference. Furthermore, substrate recognition by ADAM10 in general is incompletely understood. One possible explanation is that conformational arrangements in the GPR37 ectodomain enable favorable proximity between the cleavage site and the ADAM10 metalloprotease domain. Moreover, the possibility that the mechanism involves interaction between GPR37 and additional, as yet unidentified, regulatory proteins should be taken into consideration. Additionally, the conformation of ADAM10 could be regulated to facilitate GPR37 cleavage. For example, interaction between ADAM10 and specific members of TspanC8 tetraspanins (Matthews et al., 2017; Saint-Pol et al., 2017) might help to position the metalloprotease domain in a favorable orientation to cleave GPR37. It is also possible that GPR37 and the cleaving enzyme might locate in adjacent cells instead of the same cell. Although examples of ADAM10 substrates being cleaved in trans are rare, this kind of uncommon cleavage mechanism has been shown for ephrin A5 (Janes et al., 2005). Proprotein convertases represent another family of proteases that are commonly involved in limited proteolysis, although only a few examples of GPCR substrates exist. These include PAR1, PAR2 and a couple of adhesion family receptors (Fukuzawa & Hirose, 2006; Kim et al., 2015; Moriguchi et al., 2004; Sachan et al., 2019). In the present study, the proprotein convertase furin was found to cleave GPR37 at the Arg54↓Asp55 site. Although the N-terminal domain of GPR37 contains other potential cleavage sites for proprotein convertases, only this highly conserved site appears to be used. The finding was confirmed by site- directed mutagenesis, inhibitor-based assays and the use of furin-deficient LoVo cells. Our results also imply that the cleavage at the Arg54↓Asp55 site occurs in the TGN that is the predominant subcellular site for furin expression (Molloy et al., 1994; Thomas, 2002). This conclusion is supported by the finding that only the membrane permeable, but not the membrane impermeable, proprotein convertase inhibitor was able to inhibit receptor cleavage. Confocal microscopy also revealed that GPR37 is found in the full-length form in the secretory pathway until the TGN. The cleavage at the Arg54↓Asp55 site, however, does not appear to be as efficient as the Glu167↓Gln168 cleavage. This might be a consequence of more controlled regulation of the cleavage. For example, post-translational modifications, such as glycosylation, may affect the proteolytic processing of GPR37, or the cleavage might be triggered by some external stimuli. Additionally, it remains to be

95 investigated if there are tissue or cell type specific differences affecting the Arg54↓Asp55 cleavage of GPR37. As proprotein convertases typically process prodomains in proteins, it is possible that the first 54 amino acids in the ectodomain of GPR37 have a similar function. Thus, this peptide sequence could facilitate protein folding or inhibit the functional activity of GPR37 in a manner that is typical for prodomains in other membrane receptors (Thomas, 2002).

6.3 Functional significance of GPR37 cleavage

The finding that GPR37 is cleaved in various cell lines of distinct tissue origin and also in mouse brain implies that the proteolytic processing of the receptor is of physiological relevance. Therefore, it is intriguing to speculate on the potential functional role of GPR37 cleavage. N-terminal cleavage often affects the activation and/or signaling mechanism of GPCRs. This is particularly true for PARs and adhesion family receptors that are activated by a tethered agonist following receptor cleavage (Hamilton & Trejo, 2017; Purcell & Hall, 2018). It is tempting to hypothesize that GPR37 might be activated by a similar mechanism. However, secondary structure predictions using internet-based prediction tools (http://expasy .org/tools/) suggest that the N-terminal domain of GPR37 is mostly disordered, and thus differs from the highly structured N-terminal domain architecture of adhesion receptors. There are also differences between PARs and adhesion receptors in terms of the N-terminal domain structure and activation mechanism. PARs display longer stalk regions adjacent to the cleavage sites and the tethered agonist sequences are less hydrophobic than those of adhesion receptors (Vizurraga et al., 2020). PARs can also be activated by tethered agonists of various lengths depending on the cleaving enzyme and its cleavage site (Heuberger & Schuepbach, 2019). In contrast, adhesion receptors are cleaved specifically at the autoproteolytic site. Additionally, adhesion receptors can be allosterically activated or inhibited by external ligands binding to the N-terminal domain that is not removed by proteolytic cleavage (Vizurraga et al., 2020). Thus, the activation mechanism of GPR37 may also have its own unique features that distinguish the receptor from PARs and adhesion receptors. One possibility is that prosaposin or some other suggested ligand(s) of GPR37 could serve as an allosteric modulator(s), whereas the tethered agonist interacts with the orthosteric binding site. That could also explain the controversial results obtained by different research groups when aiming to assess the role of prosaposin in the signaling mechanism of GPR37 (Meyer et al., 2013; Nguyen et al., 2020; Yang et al., 2016).

96 The tethered agonist hypothesis is not the only option to explain the potential functional role of GPR37 cleavage. It is also possible, that the shed N-terminal fragments of GPR37 act as ligands for the receptor. Alternatively, the released N- terminal peptides may bind and functionally regulate some other cell surface receptors in the same or adjacent cells. Another possibility is that the N-terminal cleavage serves as a mechanism to desensitize or downregulate GPR37 activity. This could either follow stimulation by a yet unidentified agonist or provide means to inactivate constitutively active GPR37. For example, the melanocortin-4 receptor shows constitutive activity that is maintained by residues in its N-terminal domain (Ersoy et al., 2012), and the N-terminal tail of full-length GPR37 could have a similar function. In the case of the β1-adrenergic receptor, proteolytic cleavage of the receptor occurs in an activation-dependent manner, and mutations abrogating the Arg31↓Leu32 cleavage reduce the rate of receptor turnover (Hakalahti et al., 2010). Thus, proteolytic processing of some GPCRs is likely to play a role in receptor desensitization rather than activation. However, as GPR37 resides at the cell surface mainly in the cleaved form, it is more likely that the cleaved receptor represents the functionally active species. Yet another possibility is that GPR37 is activated by proteolytic cleavage at one site, whereas another cleavage step is required to inactivate the receptor. This kind of mechanism has been demonstrated for PARs that can be inactivated by proteolytic removal of the tethered peptide agonist (Heuberger & Schuepbach, 2019). In the case of GPR37, receptor inactivation could involve the third cleavage site close to the TM1 that was not specifically identified in this study. GPR37 may also be a genuine orphan receptor without an endogenous ligand. As mentioned in chapter 2.2.2, some of the functionally important residues, which are highly conserved among Class A GPCRs and participate in receptor activation, are missing from GPR37. Thus, GPR37 may have a functional role in cells that is independent of ligand binding. For, example GPR37 could modulate the function and trafficking of other GPCRs or receptors by dimerization. Especially D2R and

A2AR, two GPCRs that are known to interact with GPR37 (Dunham et al., 2009; Hertz et al., 2019; Morato et al., 2017; Sokolina et al., 2017), could be regulated by this kind of functional interplay. As GPR37 has been reported to interact with the dopamine transporter (Marazziti et al., 2007) and be involved in dopaminergic neurotransmission (Imai et al., 2007; Marazziti et al., 2011; Marazziti et al., 2004; Marazziti et al., 2007; X. Zhang et al., 2020), it can be speculated that receptor cleavage might also relate to the interaction between these two proteins. However,

97 it is evident that further research is required to clarify if any of the above-mentioned theories is accurate for GPR37 While toxic intracellular accumulation of GPR37 has been reported in Parkinson’s disease, recent research indicates that properly folded and trafficked GPR37 actually plays a protective role in the nervous system (B. Liu et al., 2018; Lundius, Stroth, Vukojević, Terenius, & Svenningsson, 2013; Lundius et al., 2014; Meyer et al., 2013). Therefore, the discovery that the N-terminally truncated receptor appears to be the predominant GPR37 species at the cell surface might be of great importance in order to understand the physiological function of GPR37 in health and disease. As mentioned above, the findings of the present study do not support the idea that GPR37 is prone to misfolding under stable expression conditions. Instead, GPR37 might be exceptionally sensitive to cellular disturbances such as oxidative stress, exposure to toxins or mechanical damage. This in turn could cause receptor aggregation upon certain pathological conditions such as Parkinson’s disease. It is also tempting to speculate that the proteolytic processing of GPR37 might be compromised in Parkinson’s disease and other neurological disorders associated with the receptor. Thus, further investigation is needed to determine whether the pattern of GPR37 cleavage and shedding of the N-terminal receptor peptides is altered in these diseases. It also remains unclear at present if ubiquitination by Parkin or interaction with other proteins, such as D2R and A2AR, could contribute to the disease mechanism. As GPR37 is implicated in oligodendrocyte differentiation and myelination, it could be hypothesized that proteolytic processing of the receptor plays a role in GPR37 activity also in oligodendrocytes. Interestingly, prosaposin mutations in human patients have been reported to cause loss of myelin and mature oligodendrocytes (Hulkova et al., 2001). Thus, the mechanism may also involve interaction between GPR37 and prosaposin.

98 7 Conclusions

GPR37 is an orphan GPCR that is highly expressed in the brain and has been linked to some vital physiological processes in health and disease. However, the basic biochemical behavior of the receptor and the mechanisms of its regulation are poorly characterized. As GPR37 has been reported to accumulate in the brains of Parkinson’s disease patients and researchers have observed poor plasma membrane expression of the receptor upon transient overexpression, GPR37 has been described as a GPCR that folds inefficiently and tends to aggregate inside the cells. In the present study, the biosynthetic profile and proteolytic processing of GPR37 were investigated in a heterologous expression system. The results revealed a novel mechanism to modulate GPR37 at the post-translational level. The key findings of this study are summarized as follows: 1. GPR37 folds and matures efficiently in a stable expression system, and a significant number of receptors is trafficked to the plasma membrane. However, the N-terminal domain of GPR37 is subject to proteolytic processing at Arg54↓Asp55, at Glu167↓Gln168 and a third site most probably located close to the first TM domain. 2. The cleaved GPR37 ectodomain is shed from cells and the remaining cleaved membrane-bound receptor species is the predominant receptor form found at the cell surface. 3. The main GPR37 cleaving enzyme at the Glu167↓Gln168 site is the metalloprotease ADAM10. GPR37 is additionally processed by the proprotein convertase furin at the Arg54↓Asp55 site. 4. ADAM10 is a physiologically relevant GPR37 cleaving enzyme that processes the receptor in mouse brain tissue in addition to the heterologous cell models. These results identify GPR37 as the first GPCR that is a substrate of ADAM10 and one of the rare examples that is processed by furin. As limited proteolysis of GPCRs is still a relatively poorly understood concept, ADAMs and proprotein convertases may turn out to be more commonly involved in the processing of GPCRs. Considering the importance of N-terminal cleavage in the activation mechanism of some GPCRs together with our finding that GPR37 exists at the cell surface mainly in the cleaved form, GPR37 processing is likely to play an important role in the functional activity of the receptor. In conclusion, these findings provide novel information about the proteolytic mechanisms involved in GPCR processing in general and will help to determine the full potential of GPR37 as a therapeutic target.

99

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124 Yoshida, M., Miyazato, M., & Kangawa, K. (2012). Orphan GPCRs and methods for identifying their ligands. Methods Enzymol, 514, 33-44. doi:10.1016/B978-0-12- 381272-8.00002-7 Young, B., Wertman, J., & Dupre, D. J. (2015). Regulation of GPCR Anterograde Trafficking by Molecular Chaperones and Motifs. Prog Mol Biol Transl Sci, 132, 289- 305. doi:10.1016/bs.pmbts.2015.02.012 Zeng, Z., Su, K., Kyaw, H., & Li, Y. (1997). A novel endothelin receptor type-B-like gene enriched in the brain. Biochem Biophys Res Commun, 233(2), 559-567. doi:10.1006/bbrc.1997.6408 Zhang, M. X., & Wu, G. Y. (2019). Mechanisms of the anterograde trafficking of GPCRs: Regulation of AT1R transport by interacting proteins and motifs. Traffic, 20(2), 110- 120. doi:10.1111/tra.12624 Zhang, X., Mantas, I., Fridjonsdottir, E., Andren, P. E., Chergui, K., & Svenningsson, P. (2020). Deficits in Motor Performance, Neurotransmitters and Synaptic Plasticity in Elderly and Experimental Parkinsonian Mice Lacking GPR37. Front Aging Neurosci, 12, 84. doi:10.3389/fnagi.2020.00084 Zhang, Y., Chen, K., Sloan, S. A., Bennett, M. L., Scholze, A. R., O'Keeffe, S., . . . Wu, J. Q. (2014). An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci, 34(36), 11929-11947. doi:10.1523/JNEUROSCI.1860-14.2014 Zheng, W., Zhou, J., Luan, Y., Yang, J., Ge, Y., Wang, M., . . . Chen, J. F. (2018). Spatiotemporal Control of GPR37 Signaling and Its Behavioral Effects by Optogenetics. Front Mol Neurosci, 11, 95. doi:10.3389/fnmol.2018.00095 Zhou, Q., Yang, D., Wu, M., Guo, Y., Guo, W., Zhong, L., . . . Zhao, S. (2019). Common activation mechanism of class A GPCRs. Elife, 8. doi:10.7554/eLife.50279

125

126 Original publications

I Mattila, S. O., Tuusa, J. T., & Petäjä-Repo, U. E. (2016) The Parkinson’s disease- associated receptor GPR37 undergoes metalloprotease-mediated N-terminal cleavage and ectodomain shedding. Journal of Cell Science, 129(7), 1366-1377. doi: 10.1242/jcs.176115 II Mattila, S. O., Tuhkanen, H. E., Lackman, J. J., Konzack, A., Morató, X., Argerich, J., Saftig, P., Ciruela, F., & Petäjä-Repo, U. E. (2020) GPR37 is processed in the N- terminal ectodomain by ADAM10 and furin. The FASEB Journal, Under revision Reprinted with permission from the Journal of Cell Science © The Company of Biologists (I).

Original publications are not included in the electronic version of the dissertation.

127

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1594. Daddali, Ravindra (2020) Molecular mechanisms regulating the onset of labor : comparative proteomics and miRNAomics of human spontaneous and elective labors 1595. Niemi-Nikkola, Ville (2021) Spinal fractures and spinal cord injuries : incidence, epidemiological characteristics and survival 1596. Nurkkala, Marjukka (2021) Häiriintynyt syömiskäyttäytyminen, paino ja fyysinen aktiivisuus : nuoret miehet ja ylipainoiset työikäiset 1597. Eteläinen, Sanna (2021) Gestational diabetes : screening, diagnosis and consequences 1598. Roininen, Nelli (2021) New prognostic markers and prognosis in various breast cancer subtypes 1599. Hyrkäs-Palmu, Henna (2021) Cold weather-related sensitivity among asthmatics and determinants affecting it 1600. Haukilahti, Anette (2021) Sudden cardiac death, cardiac mortality, and morbidity in women : electrocardiographic risk markers 1601. Lukkari, Sari (2021) Association between perinatal and childhood risk factors with mental disorders in adolescence 1602. Karjula, Salla (2021) Long-term consequences of polycystic ovary syndrome on mental health and health-related quality of life 1603. Kinnunen, Lotta (2021) The association of parental somatic illness with offspring’s mental health 1604. Jaurila, Henna (2021) Tissue repair and metabolic dysfunction in sepsis 1605. Kiviahde, Heikki (2021) Occlusal characteristics in Northern Finland Birth Cohort (NFBC) 1966 subjects : a longitudinal study 1606. Fouda, Shaimaa (2021) Studies on tooth loss, mandibular morphology and dental prostheses : clinical, radiological and in vitro examinations 1607. Leinonen, Anna-Maiju (2021) Technology for promoting physical activity in young men 1608. Lingaiah, Shilpa (2021) Markers assessing bone and metabolic health in polycystic ovary syndrome 1609. Ukkola, Leila (2021) Potilaan informointi ionisoivalle säteilylle altistavien kuvantamistutkimusten yhteydessä

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