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Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function

Hsiao, C.-C.

Publication date 2015 Document Version Final published version

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Citation for published version (APA): Hsiao, C-C. (2015). Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function.

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Download date:26 Sep 2021 1 Introduction Published in part in Wikipedia Wikipedia (http://www.wikipedia.org/) is a web-based, free-content encyclopedia and uses an openly editable model. Since its launch in 2001, Wikipedia has become the most popular reference site and source of information worldwide. Dr. Martin Stacey started to generate articles of adhesion GPCRs in Wikipedia in collaboration with the Adhesion GPCR Consortium (www.adhesiongpcr.org). Parts of the introduction of this thesis will be published at Wikipedia and shared in this way with the research community interested in adhesion GPCRs. Introduction

The biological term “cell” has been introduced by Robert Hooke in 1665. In 1838, Matthias Schleiden and Theodor Schwann in 1838 postulated the “Cell theory”, saying that all living 1 organisms are made of one or more cells, which is the most basic module in life1. The number of cells in the human body has been estimated as about 37 trillion2. Communication among these huge numbers of cells is required for the proper developing and functioning of the organism, and accordingly, failures in cell communication are at the basis of a large variety of diseases. Cells correspond with their environment through receptors that translate extracellular signals into intracellular messages. Seven-transmembrane (7TM) receptors are the largest superfamily of receptors in the human genome3. 7TM receptors trigger signaling through activation of heterotrimeric guanine nucleotide-binding (G proteins); therefore, they are often called G -coupled receptors (GPCRs)3. GPCRs are activated by various ligands including biogenic amines, amino acids, ions, lipid, peptides, proteins, and other molecules. They control almost all types of physiological functions and have been implicated in numerous diseases4. Notably, about 50% of all modern therapeutic drugs target GPCRs. In 1994 (Alfred Gilman and Martin Rodbell) and 2012 (Robert Lefkowitz and Brian Kobilka), Nobel Prizes have been awarded to work on GPCRs and G protein-associated signaling. Based on phylogenetic comparison of the 7TM regions, GPCRs have been grouped into five main families: Glutamate, , Adhesion, /Taste 2, and Secretin (GRAFS classification system)5,6. This thesis focuses on the study of adhesion class GPCRs, the most recently discovered and hitherto most poorly understood family of GPCRs7. In this Introduction, I summarize current knowledge about their structure, ligands, signaling, and functions in health and disease.

Adhesion class G protein-coupled receptors (adhesion GPCRs) Adhesion GPCRs are large noncanonical GPCRs with a remarkable structure. Most adhesion GPCRs possess a highly conserved cysteine-rich juxtamembranous GPCR proteolysis site (GPS) of ~50 amino acids that facilitates autocatalytic processing into an extracellular N-terminal fragment (NTF) and a 7TM/cytoplasmic C-terminal fragment (CTF)8-11 (Fig. 1). Recently, it became clear that the GPS is part of a much larger ~320-residue GPCR autoproteolysis-inducing (GAIN) domain that facilitates subsequent non-covalent association between the NTF and the CTF9 giving rise to the characteristic bipartite structure of adhesion GPCRs12,13. GPS proteolysis has been implied to be important for the maturation, stability, trafficking, and function of the receptors14. Chapters 3-5 of this thesis provide additional evidence in support of this idea for the adhesion GPCRs CD97 and GPR56. However, recent studies show that for some adhesion GPCRs, the sequence of GPS motif rather than the proteolytic event is probably essential for receptor signaling and function15,16. Therefore, posttranslational modifications that affect proteolysis remain to be identified. Chapter 2 shows that N-glycosylation of the NTF of CD97 modulates GPS proteolysis. Despite their wide distribution and some remarkable phenotypes resulting from lack or gain of function, adhesion GPCRs have been ‘functional orphans’ for a long time7. Only recently, concepts started to develop about how these non-canonical GPCRs are activated17.

9 C H C x C x x x N‐terminal x Extracellular N‐terminal H Extracellular Fragment (NTF) C T domain (ECD) Fragment (NTF) x domain (ECD) GAIN T x x GAIN GPS x GPS x C‐terminal 7TM domain C‐terminal 7TM domain Fragment (CTF) Intracellular Fragment (CTF) domain (ICD) Intracellular domain (ICD)

Fig. 1. Schematic structure of adhesion GPCRs Molecular design and terminology of adhesion GPCRs based on cleavage (left) and topology (right). Adhesion GPCRs consist of an extracellular domain, a 7TM domain, and an intracellular domain. The GAIN domain is a specific protein fold that allows autoproteolysis at a GPS motif and subsequent association of the cleaved N- and C-terminal fragments. Adhesion GPCR ligands (●) interact with the extracellular domain. Figure based on ref. 13

However, neither the role of the identified binding partners nor the cooperation between the NTF and the CTF of adhesion GPCRs is well understood yet16. The NTF is responsible for the unusually large size of most adhesion GPCRs7. The NTF bears protein domains, such as cadherin, epidermal growth factor (EGF), immunoglobulin, and leucin-rich repeats, which are able to mediate contacts with cellular or matrix-associated molecules (Fig. 2). About a dozen binding partners have been identified so far. Notably, these binding partners are structurally highly diverse and have been assigned to only a few adhesion GPCRs7. The identified-binding partners of adhesion GPCRs are cellular or matricellular molecules and not – as in other GPCR families – small molecules or peptides7. Furthermore, binding partners are not only found on opposing cells or in the extracellular matrix, but also on the surface of the same cell, and adhesion GPCRs can associate with multiple partners7. Adhesion GPCRs have been confirmed recently to mediate G protein signaling7. For instance,

GPR56 was found to associate with Gαq/11 and Gα12/13 using immunoprecipitation assays 18,19 and dominant-negative G protein-constructs . Loss of Gα12/13 or GPR56 in mice caused comparable malformations of the brain suggesting that GPR56 indeed mediates activation 20,21 of Gα12/13 . In addition, cyclic adenosine monophosphate (cAMP) and inositol phosphate (IP3) accumulation assays in overexpressing cells demonstrated that other adhesion GPCRs, 22 such as EMR2 and GPR97, couple to Gα15 and Gαo proteins . Next to activating G proteins, adhesion GPCRs can engage G protein-independent signaling pathways. The Rho family of small GTPases affects development, cytoskeleton dynamics, cell migration, and other cellular functions23. GPR56 antibody-crosslink and ligation by collagen III has been shown to activate the RhoA signaling pathway19,24. Moreover, GPR56 can recruit β-, which mediates receptor endocytosis, ubiquitylation, and non-G-protein signaling25. Canonical GPCRs are tightly controlled by desensitization via the GPCR kinase (GRK)-β-arrestin system. Activated receptors are phosphorylated intracellularly by GRKs to facilitate interaction with β-, which inhibit further binding of G proteins and cause receptor internalization3. Phorbol 12-

10 Introduction myristate 13-acetate (PMA), an activator of protein kinase C (PKC), downregulates CD97 and GPR56 on tumor cells18,26. PKC can phosphorylate uncoupled GPCRs directly or activate 1 GRKs to promote rapid internalization and desensitization3. The more than 30 adhesion GPCRs facilitate cell adhesion, orientation, migration, and positioning7. Studies in C. elegans and D. melanogaster have demonstrated essential roles in planar cell and tissue polarity, and in neuronal development. Studies in vertebrates provided evidence for involvement in various developmental processes, in immunity, and in tumorigenesis. Since the adhesion GPCR nomenclature has been highly diverse, members of the Adhesion GPCR Consortium (www.adhesiongpcr.org) recently developed a new nomenclature published in 2015 in Pharmacological Reviews7. This nomenclature subdivides the 33 human adhesion GPCRs based on phylogenetic comparison of their 7TM domains into nine subfamilies: subfamilies ADGRL (), ADGRA, ADGRC (CELSRs), ADGRD, ADGRG, and ADGRV are evolutionary old, while the subfamilies ADGRE (EGF-TM7), ADGRF, and ADGRB (BAIs) are only found in vertebrates (Fig. 2). The following section provides details about (members of) the subfamilies ADGRE (EGF-TM7) and ADGRG and in particular about the two adhesion GPCRs studied in this thesis: CD97 and GPR56.

Fig. 2. Adhesion GPCRs can be subdivided into nine subfamilies with 33 mammalian homologs Each subfamily has its own unique extracellular folds within the NTF (bracketed domains cannot be found in every member of the family) and high similarity at the 7TM domain. GPR123 does not have a GAIN domain. Names according to the new nomenclature are provided next to the traditional names. Figure modified from ref. 13

11 Family II (EGF-TM7; ADGRE) The EGF-TM7 family of adhesion GPCRs is expressed mainly in leukocytes and comprises five members: EGF-like module containing mucin-like receptor 1 (EMR1; ADGRE1), EMR2 (ADGRE2), EMR3 (ADGRE3), EMR4 (ADGRE4), and CD97 (ADGRE5)7. EGF-TM7 receptors possess variable numbers of EGF-like domains resulting from alternative mRNA splicing27. Functionally, EGF-TM7 receptors are involved in leukocyte development and activation. CD97 – one of the most-studied adhesion GPCRs – is found to play multiple roles, not only in immunology but also in tumorigenesis.

EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1, F4/80; ADGRE1) EMR1 expression in human is restricted to eosinophils and is a specific marker for these cells28. Interestingly, the murine homolog of EMR1, F4/80 is a well-known and widely-used marker of murine macrophage populations29. The NTF of EMR1 contains 4-6 EGF-like domains in human and 4-7 EGF-like domains in the mouse27.

Function Utilizing F4/80 knockout mice, Lin et al. showed that F4/80 is not necessary for the development of tissue macrophages but is required for the induction of efferent CD8+ regulatory T cells needed for peripheral tolerance30.

Clinical significance Legrand et al. recently demonstrated that EMR1 can serve as a therapeutic target for depletion of these cells in eosinophilic disorders by using afucosylated antibodies31.

EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2, CD312; ADGRE2) EMR2 is expressed by monocytes/macrophages, dendritic cells and all types of granulocytes32. EMR2 is closely related to CD97 with 97% amino-acid identity in the EGF-like domains. The NTF of EMR2 presents 2-5 EGF-like domains in human27. Mice lack the Emr2 gene33.

Ligand Like CD97, the fourth EGF-like domain of EMR2 binds chondroitin sulfate B to mediate cell attachment34.

Signaling IP3 accumulation assays in overexpressing HEK293 cells have demonstrated coupling of 22 EMR2 to Gα15 . Further, distribution, translocation, co-localization of the NTF and CTF of EMR2 within lipid rafts may affect cell signaling35.

12 Introduction

Function The expression of EMR2 and CD97 on activated lymphocytes and myeloid cells promotes 1 binding with their ligand chondroitin sulfate B on peripheral B cells, indicating a role in leukocyte interaction36. The interaction between EMR2 and chondroitin sulfate B in inflamed rheumatoid synovial tissue suggests a role of the receptors in the recruitment and retention of leukocytes in synovium of arthritis patients37. Upon neutrophil activation, EMR2 rapidly moves to membrane ruffles and the leading edge of the cell. Additionally, ligation of EMR2 by antibody promotes neutrophil and macrophage effector functions suggesting a role in potentiating inflammatory responses35,38.

Clinical significance EMR2 is rarely expressed by tumor cell lines and tumors, but has been found on breast and colorectal adenocarcinoma39,40. In breast cancer, robust expression and different distribution of EMR2 is inversely correlated with survival41.

EGF-like module-containing mucin-like hormone receptor-like 3 (EMR3; ADGRE3) EMR3 expression is restricted to monocytes/macrophages, myeloid dendritic cells, and mature granulocytes in human42. Interestingly, transcription of the EMR3 results in two alternative spliced forms: a surface protein with extracellular, 7TM, and intracellular domains as well as a truncated soluble form of only the extracellular domain43. Mice, next to Emr2, lack the Emr3 gene33.

Ligands A potential ligand of EMR3 likely is expressed on human macrophage and activated neutrophils43.

EGF-like module-containing mucin-like hormone receptor-like 4 (EMR4, GPR127, FIRE; ADGRE4) EMR4 expression is expressed by monocytes/macrophages and CD8– dendritic cell subsets in the mouse, but is inactivated in human due to a single-nucleotide deletion44. The NTF of EMR4 contains 2 EGF-like domains in mouse27.

Ligands A potential ligand of mouse EMR4 is found on the B lymphoma cell line A2044.

Cluster of differentiation 97 (CD97, BL-Ac[F2]; ADGRE5) CD97 is widely expressed on, among others, hematopoietic stem and progenitor cells, immune cells, epithelial cells, muscle cells as well as their malignant counterparts45-50. The N-terminal fragment of CD97 contains 3-5 EGF-like domains in human and 3-4 EGF-like domains in mice27.

13 Ligands CD55 interacts with the first and second EGF-like domains of CD9751, chondroitin sulfate B with the fourth EGF-like domain52, α5β1 and αvβ3 integrins with an RGD downstream the EGF-like domains53; and CD90 (Thy-1) with the GAIN domain54. N-glycosylation of CD97 within the EGF domains is crucial for CD55 binding55.

Signaling Transgenic expression of a CD97 in mice enhanced levels of nonphosphorylated membrane- bound β-catenin and phosphorylated Akt56. Furthermore, ectopic CD97 expression facilitated

RhoA activation through binding of Gα12/13 as well as induced Ki67 expression and phosphorylated ERK and Akt through enhancing receptor 1 (LPAR1) signaling57,58. Lysophosphatidylethanolamine (LPE; a plasma membrane component) and lysophosphatidic

acid (LPA) use heterodimeric LPAR1–CD97 to drive Gi/o protein–phospholipase C–inositol 1,4,5-trisphosphate signaling and induce [Ca2+] in breast cancer cells59.

Function In the immune system, CD97 is known as a critical mediator of host defense. Upon lymphoid, myeloid cells and neutrophil activation, CD97 is upregulated to promote adhesion and migration to sites of inflammation60. Moreover, it has been shown that CD97 regulates granulocyte homeostasis. Mice lacking CD97 or its ligand CD55 have twice as many granulocytes as wild-type mice possibly due to enhanced granulopoiesis61. Antibodies against CD97 have been demonstrated to diminish various inflammatory disorders by depleting granulocytes62. Notably, CD97 antibody-mediated granulocytopenia only happens under the condition of pro-inflammation via an Fc receptor-associated mechanism63. Finally, the interaction between CD97 and its ligand CD55 regulates T-cell activation and increases proliferation and cytokine production64,65. Changes in the expression of CD97 have been described for auto-inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. The expression of CD97 on macrophage and the abundant presence of its ligand CD55 on fibroblast-like synovial cells suggest that the CD97-CD55 interaction is involved in the recruitment and/or retention of macrophages into the synovial tissue in rheumatoid arthritis66. CD97 antibodies and lack of CD97 or CD55 in mice reduced synovial inflammation and joint damage in collagen- and K/BxN serum transfer-induced arthritis67,68. In brain tissue, CD97 is undetectable in normal white matter, and expression of CD55 is fairly restricted to the endothelium. In pre-active lesion, increased expression of CD55 in endothelial cells and robust CD97 expression on infiltrating leukocytes suggest a possible role of both molecules in immune cell migration through the blood-brain barrier69. Additionally, soluble NTFs of CD97 are detectable in the serum of patients with rheumatoid arthritis and multiple sclerosis66. Outside the immune system, CD97 is likely involved in cell–cell interactions. CD97 in colonic enterocytes strengthens E-cadherin-based adherens junctions to maintain lateral

14 Introduction cell-cell contacts and regulates the localization and degradation of β-catenin through glycogen synthase kinase-3β (GSK-3β) and Akt signaling56. Chapter 3 of the thesis reveals 1 that ectopic CD97 expression upregulates the expression of N-cadherin and β-catenin in HT1080 fibrosarcoma cells leading to enhanced cell-cell aggregation. CD97 is expressed at the sarcoplasmic reticulum and the peripheral sarcolemma in skeletal muscle. However, lack of CD97 only affects the structure of the sarcoplasmic reticulum, but not the function of skeletal muscle50. In addition, CD97 promotes angiogenesis of the endothelium through to α5β1 and αvβ3 integrins, which contributes to cell attachment53.

Clinical significance CD97 expression in cancer was first reported for dedifferentiated thyroid carcinoma and their lymph node metastases26. CD97 is expressed on many types of tumors including thyroid, gastric, pancreatic, esophageal, colorectal, and oral squamous carcinomas as well as glioblastoma and glioblastoma-initiating cells26,40,70-74. In addition, enhanced CD97 expression has been found at the invasion front of tumors75, suggesting a possible role in tumor migration/invasion72,75, and correlated with a poorer clinical prognosis70,71,73,76,77. Interestingly, CD97 has isoform- specific functions in some tumors. For instance, the small EGF(1,2,5) isoform promoted tumor invasion and metastasis in gastric carcinoma78; the small EGF(1,2,5) isoform induced but the full length EGF(1-5) isoform suppressed gastric carcinoma invasion79. Forced CD97 expression induced cell migration, activated proteolytic matrix metalloproteinases (MMPs), and enhanced secretion of the chemokines interleukin (IL)-880. Tumor suppressor microRNA- 126, often downregulated in cancer, was found to target CD97 thereby modulating cancer progression81. CD97 can heterodimerize with the LPAR1, a canonical GPCR that is implied in tumor progression, to modulate synergistic functions and LPA-mediated Rho signaling57,58. It has been shown that CD97 regulates localization and degradation of β-catenin56. GSK-3β, inhibited in some cancer, regulates the stability of β-catenin in cytoplasm and subsequently, cytosolic β-catenin moves into the nucleus to facilitate expression of pro-oncogenic genes82,83. Because of its role in tumor invasion and angiogenesis, CD97 is a potential therapeutic target. Several treatments reduce CD97 expression in tumor cells such as cytokine tumor growth factor (TGF)β as well as the compounds sodium butyrate, retinoic acid, and troglitazone70,71,84. Taken together, experimental evidence indicates that CD97 plays multiple roles in tumor progress. The thesis includes new studies on CD97 in cell aggregation (Chapter 3), migration/ invasion (Chapter 4), and apoptosis (Chapter 5).

Family VIII (ADGRG) Subfamily VIII of adhesion GPCRs comprises seven members: G protein-coupled receptor 56 (GPR56; ADGRG1), GPR64 (ADGRG2), GPR97 (ADGRG3), GPR112 (ADGRG4), GPR114 (ADGRG5), GPR126 (ADGRG6), and GPR128 (ADGRG7)7.

15 16

Table 1. Family II of adhesion GPCRs

Receptor EMR1 EMR2 EMR3 EMR4 CD97

Structure

Expression Human Eosinophils Monocytes Monocytes Gene likely inactive All leukocytes Macrophages Macrophages Non-immune cells Myeloid dendritic cells Myeloid dendritic cells Tumor cells Granulocytes Mature granulocytes Few tumor cells Mouse Monocytes Gene absent Gene absent Monocytes All leukocytes Macrophages Macrophage subsets Non-immune cells Myeloid dendritic cells CD8– dendritic cells Eosinophils EGF domains Human 4-6 2-5 2 - 3-5 Mouse 4-7 - - 2 3-4 Ligands Unknown Chondroitin sulfate B Unknown Unknown CD55 Chondroitin sulfate B α5β1 and αvβ3 integrins CD90

Signaling Unknown Gα15 Unknown Unknown Gα12/13 (RhoA, ERK, Akt) Heterodimeric LPAR1–CD97 Function Peripheral immune tolerance Neutrophil activation and Unknown Unknown Granulopoiesis migration T cell recruitment/activation Angiogenesis Clinical Therapeutic target for Express on tumor Unknown Unknown Tumorigenesis Significance eosinophilic disorders

Triangles indicate EGF-like domains; circle means cleavage-deficient GPS motif; half circles indicate autoproteolytic cleavage of the receptor at the GPS motif imbedded within the GAIN domain. Table modified from ref. 25 Introduction

G protein-coupled receptor 56 (GPR56, TM7XN1; ADGRG1) GPR56 is expressed in liver, muscle, neural, and cytotoxic lymphoid cells in human as well 1 as in hematopoietic precursor, muscle, and developing neural cells in the mouse7.

Ligands GPR56 binds transglutaminase 2 to suppress tumor metastasis85 and binds collagen III to regulate cortical development and lamination24.

Signaling 18 GPR56 couples to Gαq/11 protein upon association with the CD9 and CD81 . Forced GPR56 expression activates NF-kB, PAI-1, and TCF transcriptional response elements86. The splicing of GPR56 induces tumorigenic responses as a result of activating 87 transcription factors, such as COX2, iNOS, and VEGF . GPR56 couples to the Gα12/13 protein and activates RhoA and mammalian target of rapamycin (mTOR) pathway upon ligand binding19,24,88,89. Lack of the NTF of GPR56 causes stronger RhoA signaling and β-arrestin accumulation, leading to extensive ubiquitination of the CTF25. Finally, GPR56 suppresses PKCα activation to regulate angiogenesis90.

Function GPR56 was the first adhesion GPCR causally linked to a disease. Loss-of-function mutations in GPR56 cause a severe cortical malformation known as bilateral frontoparietal polymicrogyria (BFPP)91-97 (Fig. 3). Investigating the pathological mechanism of disease-associated GPR56 mutations in BFPP has provided mechanistic insights into the functioning of adhesion GPCRs. Piao’s and our group demonstrated that disease- associated GPR56 mutations cause BFPP via multiple mechanisms, see Chapter 6 and98-100. Li et al. demonstrated that GPR56 regulates pial basement membrane (BM) organization during cortical development21. Disruption of the Gpr56 gene in mice leads to neuronal malformation in the cerebral cortex, which resulted in 4 critical pathological morphologies: defective pial BM, abnormal localized radial glial endfeet, malpositioned Cajal-Retzius cells, and overmigrated neurons21. Furthermore, the interaction of GPR56 and collagen III inhibits neural migration to regulate lamination of the cerebral cortex24. Next to GPR56, the α3β1 integrin is also involved in pial BM maintenance. Study from Itga3 (α3 integrin)/ Gpr56 double knockout mice showed increased neuronal overmigration compared to Gpr56 single knockout mice, indicating cooperation of GPR56 and α3β1 integrin in modulation of the development of the cerebral cortex101. More recently, the Walsh laboratory showed that alternative splicing of GPR56 regulates regional cerebral cortical patterning102. Outside the nervous system, GPR56 has been linked to muscle function and male fertility. The expression of GPR56 is upregulated during early differentiation of human myoblasts. Investigation of Gpr56 knockout mice and BFPP patients showed that GPR56 is required for in vitro myoblast fusion via signaling of serum response factor (SRF) and

17 Fig. 3. Defective GPR56 causes BFPP (A) MRI images in brains from patient with BFPP and health individual. Arrowheads show the regions affected by polymicrogyria; arrows show the central sulcus and sylvian fissure, respectively. (B) Stars show missense-mutated sites found in GPR56 with ten mutations at the NTF, three mutations in the GAIN-GPS region, and five mutations in the 7TM region. Arrows show the mutated sites for two splicing mutations (S), four deletion mutations (Δ1=1 bp, Δ3=3 bp, Δ7=7 bp), one insertion mutation (ins1=1 bp), and one deletion–insertion mutation (Δ6/ins17=6/17 bp). Figure modified from ref. 84

nuclear factor of activated T-cell (NFAT), but is not essential for muscle development in vivo103. Additionally, GPR56 is a transcriptional target of peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 and regulates overload-induced muscle hypertrophy 89 through Gα12/13 and mTOR signaling . Therefore, the study of knockout mice revealed that GPR56 is involved in testis development and male fertility104. Studies in the hematopoietic system disclosed that during endothelial to hematopoietic stem cell transition, Gpr56 is a transcriptional target of the heptad complex of hematopoietic transcription factors, and is required for hematopoietic cluster formation105. Recently, two studies showed that GPR56, is a cell autonomous regulator of oligodendrocyte development 88,106 through Gα12/13 proteins and Rho activation . Della Chiesa et al. demonstrate that GPR56 is expressed on CD56dull NK cells107. We showed that all human cytotoxic lymphocytes, including CD56dull NK cells and CD27–CD45RA+ effector-type CD8+ T cells, express GPR56108. However, many questions remained regarding the regulation and function of GPR56 in human cytotoxic lymphocytes. This thesis addresses the role of GPR56 in human NK cells in Chapter 7.

Clinical significance Mutations in GPR56 cause the brain developmental disorder BFPP, characterized by disordered cortical lamination in frontal cortex91. Mice lacking expression of GPR56

18 Introduction develop a comparable phenotype21. Furthermore, loss of GPR56 leads to reduced fertility in male mice, resulting from a defect in seminiferous tubule development104. 1 GPR56 is expressed in glioblastoma/astrocytoma86 as well as in esophageal squamous cell109, breast, colon, non-small cell lung, ovarian, and pancreatic carcinoma110. GPR56 was shown to localize together with α-actinin at the leading edge of membrane filopodia in glioblastoma cells, suggesting a role in cell adhesion/migration86. In addition, recombinant GPR56-NTF protein interacts with glioma cells to inhibit cellular adhesion86. Inactivation of Von Hippel-Lindau (VHL) tumor-suppressor gene and hypoxia suppressed GPR56 in a renal cell carcinoma cell line, but hypoxia influenced GPR56 expression in breast or bladder cancer cell lines111. GPR56 is a target gene for vezatin, an adherens junctions transmembrane protein, which is a tumor suppressor in gastric cancer112. Xu et al. used an in vivo metastatic model of human melanoma to show that GPR56 is downregulated in highly metastatic cells85. Later, by ectopic expression and RNA interference they confirmed that GPR56 inhibits melanoma tumor growth and metastasis. Silenced expression of GPR56 in HeLa cells enhanced apoptosis and anoikis, but suppressed anchorage-independent growth and cell adhesion110. High ecotropic viral integration site-1 acute myeloid leukemia (EVI1-high AML) expresses GPR56 that was found to be a transcriptional target of EVI1113. Silencing expression of GPR56 decreases adhesion, cell growth and induces apoptosis through reduced RhoA signaling. GPR56 suppresses the angiogenesis and melanoma growth through inhibition of vascular endothelial growth factor (VEGF) via PKCα signaling pathway90. Furthermore, GPR56 expression was found to be negatively correlated with the malignancy of melanomas in human patients90.

G protein-coupled receptor 64 (GPR64, HE6; ADGRG2) GPR64 is mainly expressed in human and mouse epididymis as well as human prostate and parathyroid7. GPR64, together with F-actin scaffold, locates at the nonciliated principal cells of the proximal male excurrent duct epithelia, where reabsorption of testicular fluid and concentration of sperm takes place114,115.

Function Targeting of Gpr64 in mice causes reduced fertility or infertility in males; but the reproductive capacity was unaffected in females116. Unchanged hormone expression in knockout males indicates that the receptor functions immediately in the male genital tract. Lack of Gpr64 expression causes sperm stasis and duct obstruction due to abnormal fluid reabsorption. In addition, expression of GPR64 has been found in fibroblast-like synovial cells obtained from osteoarthritis but not from rheumatoid arthritis117.

Clinical significance GPR64 is significantly overexpressed in the Wnt signaling-dependent subgroup of medulloblastoma118, as well as in ewing sarcomas and carcinomas derived from prostate,

19 kidney or lung119. Richter et al. demonstrated that GPR64 promotes tumor invasion and metastasis through placental growth factor and MMP1119.

G protein-coupled receptor 97 (GPR97, Pb99; ADGRG3) GPR97 is expressed in human granulocytes and endothelial cells of the vasculature as well as in mouse granulocytes, monocytes, macrophages, and dendritic cells7.

Signaling The IP3 accumulation, aequorin, and 35S isotope binding assays in overexpressing HEK293 22 cells have demonstrated coupling of GPR97 to Gαo protein triggering cAMP . GPR97 actives cAMP response element-binding protein (CREB), NF-κB, and small GTPases to regulate cellular functions.

Function Systemic steroid exposure is a therapy to treat a variety of medical conditions and is associated with epigenetic processes such as DNA methylation that may reflect pharmacological responses and/or side effects. GPR97 was found to be differently methylated at CpG sites in the genome of blood cells from patient under systemic steroid treatment120. GPR97 is transcribed in immune cells. Gene-deficient mice revealed that Gpr97 is crucial for maintaining B-cell population via constitutive CREB and NF-κB activities121. Human lymphatic endothelial cells (LECs) abundantly express GPR97. Silencing GPR97 in human LECs indicated that GPR97 modulates cytoskeletal rearrangement, cell adhesion and migration through regulating the small GTPase RhoA and cdc42122. In vertebrates, GPR97 has an indispensable role in the bone morphogenetic proteins (BMP) signaling pathway in bone formation. A microarray meta-analysis revealed that mouse Gpr97 is a direct transcriptional target of BMP signaling in long bone development123.

G protein-coupled receptor 112 (GPR112; ADGRG4) GPR112 is expressed in human enterochromaffin cells124 and in the mouse intestine7. The NTF of GPR112 contains pentraxin (PTX)-like modules7. GPR112 has been identified as a marker for neuroendocrine carcinoma cells124.

G protein-coupled receptor 114 (GPR114; ADGRG5) GPR114 mRNA is specifically expressed in human eosinophils as well as in mouse lymphocytes, monocytes, macrophage, and dendritic cells7.

Signaling The cAMP assay in overexpressing HEK293 cells has demonstrated coupling of GPR114 to 22 Gαs protein .

20 Introduction

G protein-coupled receptor 126 (GPR126, VIGR, DREG; ADGRG6) GPR126 is all widely expressed on stromal cells7. The N-terminal fragment of GPR126 1 contains C1r-C1s, Uegf and Bmp1 (CUB), and PTX-like modules125.

Ligand GPR126 was shown to bind collagen IV and laminin-211 promoting cAMP to mediate myelination126,127.

Signaling Upon lipopolysaccharide (LPS) or thrombin stimulation, expression of GPR126 is induced by MAP kinases in endothelial cells125. During angiogenesis, GPR126 promotes protein kinase A (PKA)–cAMP-activated signaling in endothelial cells128. Forced GPR126 expression 129 in COS-7 cells enhances cAMP levels by coupling to heterotrimeric Gαs/i proteins .

Function GPR126 has been identified in genomic regions associated with adult height, more specially trunk height130-132, pulmonary function133 and adolescent idiopathic scoliosis134. In the vertebrate nervous system, many axons are surrounded by a myelin sheath to conduct action potentials rapidly and efficiently. Applying a genetic screen in zebrafish mutants, Talbot’s group demonstrated that GPR126 affects the development of myelinated axons135. GPR126 drives the differentiation of Schwann cells through inducing cAMP levels, which causes Oct6 transcriptional activities to promote myelin gene activity136. Mutation of in zebrafish affects peripheral myelination. Interestingly, Monk’s group demonstrated domain- specific functions of GPR126 during Schwann cells development: the NTF is necessary and sufficient for axon sorting, while the CTF promotes wrapping through cAMP induction to regulate early and late stages of Schwann cells development127. Outside of neurons, GPR126 function is required for heart and inner ear development137-139. GPR126 stimulates VEGF signaling and angiogenesis by modulating VEGF receptor 2 (VEGFR2) expression through STAT5 and GATA2 in endothelial cells128.

G protein-coupled receptor 128 (GPR128; ADGRG7) GPR128 is specifically expressed in human liver as well as in mouse bone marrow and intestinal tissues7.

Function Ni et al. showed that Gpr128 deletion in mice causes reduced body weight and induced intestinal contraction frequency140.

21 Clinical significance A 111-kb amplification with breakpoints within the TRK-fused gene (a target of translocations in lymphoma and thyroid tumors) and GPR128 has been identified in the genome of patients with atypical myeloproliferative neoplasms141. Notably, the fused gene was also detected in few healthy individuals.

22 Introduction

1 GPR128 Unknown Oncologic fusion fusion Oncologic gene Bone marrow Intestine Intestinal contraction Intestinal Intestine Liver Unknown GPR126 (cAMP) s/i Collagen IV Laminin 121 Unknown Stromal cells Stromal Myelination Heart and Ear development Angiogenesis Stromal cells Stromal Gα GPR114 s Unknown Unknown Lymphocytes Monocytes Macrophage Dendritic cells Unknown Eosinophil Gα GPR112 Unknown Tumor Marker Tumor Intestine Unknown Enterochromaffin Enterochromaffin cells Unknown GPR97 (cAMP) o Unknown Unknown Granulocytes Monocytes Macrophage Dendritic cells B-cell maintenance Bone formation Granulocytes of cells Endothelial vasculature Gα GPR64 Unknown Tumor metastasis Tumor Epididymis Male fertility Male Epididymis Prostate Parathyroid Unknown GPR56 (RhoA, (with CD81/CD9) (with 12/13 q/11 Gα Collagen III TG2 BFPP suppressor Tumor Muscle Neuron cells Hematopoietic Hematopoietic stem stem Hematopoietic cell maintenance Cortical development fertility Male hypertrophy Muscle Liver Muscle Neuron lymphocytes Cytotoxic Gα mTOR) PKCα, Family VIII of adhesion GPCRs adhesion VIII of Family Signaling Pentagons indicate Cs1 and Csr/Uegf/BMP1 (CUB) domains; hexagon indicate laminin_G/PTX domains; half circles indicate autoproteolytic cleavage of the receptor the receptor of cleavage autoproteolytic indicate half circles laminin_G/PTX domains; indicate hexagon (CUB) domains; Csr/Uegf/BMP1 Cs1 and indicate Pentagons imbedded the the GPS motif within GAIN domain at Ligand Clinical Significance Mouse Structure Function Table 2. Table Receptor Expression Human

23 Scope of this thesis The autocatalytic processing of most adhesion GPCRs into two non-covalently associated fragments raises question regarding the regulation and implications of this posttranslational modification. Moreover, we just begin to understand the (patho)physiological activities of adhesion GPCRs, and studies further exploring their roles in health and disease are therefore highly warranted. This thesis focuses on the mechanism and implications of autoproteolysis of adhesion GPCRs as well as on the functions of the prototypical adhesion GPCRs CD97 and GPR56. In Chapter 2, we investigated how glycosylation affects autoproteolysis in CD97. Chapter 3 and 4 show the consequences of autoproteolytic modification for cellular functions of CD97 in cell aggregation and tumorigenesis. Furthermore, Chapter 4 provides novel insight into the role of CD97 in tumor cell migration and metastasis. Chapter 5 reports the ability of CD97 to protect cells against apoptosis. Chapter 6 describes cellular functions of GPR56 and indicates molecular mechanisms by which GPR56 mutations cause a cortical malformation, known as BFPP (bilateral frontoparietal polymicrogyria). Through studying NK cells from BFPP patients with a null mutation in the GPR56 gene and cells ectopically expressing GPR56, we explored the function of GPR56 in NK cells in Chapter 7. We found that GPR56 negatively regulates NK-cell effector functions and that its expression, driven by Hobit, declines upon cell activation. Finally, the finding of this thesis are summarized and discussed in Chapter 8.

24 Introduction

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26 Introduction

54 Wandel, E., Saalbach, A., Sittig, D., Gebhardt, 69 Visser, L. et al. Expression of the EGF-TM7 C. & Aust, G. Thy-1 (CD90) is an interacting receptor CD97 and its ligand CD55 (DAF) in partner for CD97 on activated endothelial cells. multiple sclerosis. J. Neuroimmunol. 132, 156– 1 J. Immunol. 188, 1442–1450 (2012). 163 (2002). 55 Wobus, M., Vogel, B., Schmücking, E., Hamann, 70 Steinert, M. et al. Expression and regulation J. & Aust, G. N-glycosylation of CD97 within the of CD97 in colorectal carcinoma cell lines and EGF domains is crucial for epitope accessibility in tumor tissues. Am. J. Pathol. 161, 1657–1667 normal and malignant cells as well as CD55 ligand (2002). binding. Int. J. Cancer 112, 815–822 (2004). 71 Mustafa, T. et al. Expression of the epidermal 56 Becker, S. et al. Overexpression of CD97 in growth factor seven-transmembrane member intestinal epithelial cells of transgenic mice CD97 correlates with grading and staging in attenuates colitis by strengthening adherens human oral squamous cell carcinomas. Cancer junctions. PLoS ONE 5, e8507 (2010). Epidemiol. Biomarkers Prev. 14, 108–119 (2005). 57 Ward, Y. et al. LPA receptor heterodimerizes 72 Safaee, M. et al. Overexpression of CD97 confers with CD97 to amplify LPA-initiated RHO- an invasive phenotype in glioblastoma cells and is dependent signaling and invasion in prostate associated with decreased survival of glioblastoma cancer cells. Cancer Res. 71, 7301–7311 (2011). patients. PLoS ONE 8, e62765 (2013). 58 Ward, Y. et al. CD97 amplifies LPA receptor 73 Liu, J. K. et al. Phage display discovery of novel signaling and promotes thyroid cancer progression molecular targets in glioblastoma-initiating in a mouse model. Oncogene 32, 2726–2738 (2013). cells. Cell Death Differ. 21, 1325–1339 (2014). 59 Park, S.-J. et al. Lysophosphatidylethanolamine 74 Safaee, M. et al. Proportional Upregulation utilizes LPA(1) and CD97 in MDA-MB-231 breast of CD97 Isoforms in Glioblastoma and cancer cells. Cell. Signal. 25, 2147–2154 (2013). Glioblastoma-Derived Brain Tumor Initiating 60 Leemans, J. C. et al. The epidermal growth factor- Cells. PLoS ONE 10, e0111532 (2015). seven transmembrane (EGF-TM7) receptor 75 Wobus, M., Huber, O., Hamann, J. & Aust, CD97 is required for neutrophil migration and G. CD97 overexpression in tumor cells at the host defense. J. Immunol. 172, 1125–1131 (2004). invasion front in colorectal cancer (CC) is 61 Veninga, H. et al. A novel role for CD55 in independently regulated of the canonical Wnt granulocyte homeostasis and anti-bacterial host pathway. Mol. Carcinog. 45, 881–886 (2006). defense. PLoS ONE 6, e24431 (2011). 76 Wu, J., Lei, L., Wang, S., Gu, D. & Zhang, 62 Hamann, J. et al. CD97 in leukocyte trafficking. J. Immunohistochemical expression and Adv. Exp. Med. Biol. 706, 128–137 (2010). prognostic value of CD97 and its ligand CD55 63 Veninga, H. et al. CD97 antibody depletes in primary gallbladder carcinoma. J. Biomed. granulocytes in mice under conditions of acute Biotechnol. 2012, 587672 (2012). inflammation via a Fc receptor-dependent 77 He, Z., Wu, H., Jiao, Y. & Zheng, J. Expression mechanism. J. Leukoc. Biol. 89, 413–421 (2011). and prognostic value of CD97 and its ligand 64 Capasso, M. et al. Costimulation via CD55 CD55 in pancreatic cancer. Oncol Lett 9, 793– on human CD4+ T cells mediated by CD97. J. 797 (2015). Immunol. 177, 1070–1077 (2006). 78 Liu, D. et al. The invasion and metastasis 65 Abbott, R. J. M. et al. Structural and functional promotion role of CD97 small isoform in gastric characterization of a novel T cell receptor co- carcinoma. PLoS ONE 7, e39989 (2012). regulatory protein complex, CD97-CD55. J. 79 Liu, D. et al. Role of CD97 isoforms in gastric Biol. Chem. 282, 22023–22032 (2007). carcinoma. Int. J. Oncol. 36, 1401–1408 (2010). 66 Hamann, J. et al. Expression of the activation 80 Galle, J. et al. Individual cell-based models of antigen CD97 and its ligand CD55 in rheumatoid tumor-environment interactions: Multiple synovial tissue. Arthritis Rheum. 42, 650–658 effects of CD97 on tumor invasion. Am. J. (1999). Pathol. 169, 1802–1811 (2006). 67 Kop, E. N. et al. CD97 neutralisation increases 81 Lu, Y. Y. et al. Prometastatic GPCR CD97 is a resistance to collagen-induced arthritis in mice. direct target of tumor suppressor microRNA-126. Arthritis Res. Ther. 8, R155 (2006). ACS Chem. Biol. 9, 334–338 (2014). 68 Hoek, R. M. et al. Deletion of either CD55 or 82 Wong, N. A. C. S. & Pignatelli, M. Beta-catenin- CD97 ameliorates arthritis in mouse models. -a linchpin in colorectal carcinogenesis? Am. J. Arthritis Rheum. 62, 1036–1042 (2010). Pathol. 160, 389–401 (2002).

27 83 Takahashi-Yanaga, F. & Sasaguri, T. GSK-3beta imaging studies in children with novel GPR56 regulates cyclin D1 expression: a new target for mutations: further delineation of a cobblestone- chemotherapy. Cell. Signal. 20, 581–589 (2008). like phenotype. Neurogenetics 14, 77–83 (2013). 84 Park, J.-W. et al. Troglitazone, the peroxisome 97 Santos-Silva, R. et al. Bilateral Frontoparietal proliferator-activated receptor-gamma agonist, Polymicrogyria: A Novel GPR56 Mutation and induces antiproliferation and redifferentiation an Unusual Phenotype. Neuropediatrics (2015). in human thyroid cancer cell lines. Thyroid 15, doi:10.1055/s-0034-1399754 222–231 (2005). 98 Jin, Z. et al. Disease-associated mutations affect 85 Xu, L., Begum, S., Hearn, J. D. & Hynes, R. O. GPR56 protein trafficking and cell surface GPR56, an atypical G protein-coupled receptor, expression. Hum. Mol. Genet. 16, 1972–1985 binds , TG2, and inhibits (2007). melanoma tumor growth and metastasis. Proc. 99 Ke, N. et al. Biochemical characterization of Natl. Acad. Sci. U.S.A. 103, 9023–9028 (2006). genetic mutations of GPR56 in patients with 86 Shashidhar, S. et al. GPR56 is a GPCR that is bilateral frontoparietal polymicrogyria (BFPP). overexpressed in gliomas and functions in tumor Biochem. Biophys. Res. Commun. 366, 314–320 cell adhesion. Oncogene 24, 1673–1682 (2005). (2008). 87 Kim, J.-E. et al. Splicing variants of the orphan 100 Luo, R., Jin, Z., Deng, Y., Strokes, N. & Piao, X. G-protein-coupled receptor GPR56 regulate the Disease-associated mutations prevent GPR56- activity of transcription factors associated with collagen III interaction. PLoS ONE 7, e29818 tumorigenesis. J. Cancer Res. Clin. Oncol. 136, (2012). 47–53 (2010). 101 Jeong, S.-J. et al. GPR56 functions together with 88 Ackerman, S. D., Garcia, C., Piao, X., Gutmann, α3β1 integrin in regulating cerebral cortical D. H. & Monk, K. R. The adhesion GPCR development. PLoS ONE 8, e68781 (2013). Gpr56 regulates oligodendrocyte development 102 Bae, B.-I. et al. Evolutionarily dynamic alternative via interactions with Gα12/13 and RhoA. Nat splicing of GPR56 regulates regional cerebral Commun 6, 6122 (2015). cortical patterning. Science 343, 764–768 (2014). 89 White, J. P. et al. G protein-coupled receptor 56 103 Wu, M. P. et al. G-protein coupled receptor regulates mechanical overload-induced muscle 56 promotes myoblast fusion through serum hypertrophy. Proc. Natl. Acad. Sci. U.S.A. 111, response factor- and nuclear factor of activated 15756–15761 (2014). T-cell-mediated signalling but is not essential 90 Yang, L. et al. GPR56 Regulates VEGF for muscle development in vivo. FEBS J. 280, production and angiogenesis during melanoma 6097–6113 (2013). progression. Cancer Res. 71, 5558–5568 (2011). 104 Chen, G., Yang, L., Begum, S. & Xu, L. GPR56 is 91 Piao, X. et al. G protein-coupled receptor- essential for testis development and male fertility dependent development of human frontal in mice. Dev. Dyn. 239, 3358–3367 (2010). cortex. Science 303, 2033–2036 (2004). 105 Solaimani Kartalaei, P. et al. Whole-transcriptome 92 Piao, X. et al. Genotype-phenotype analysis analysis of endothelial to hematopoietic stem cell of human frontoparietal polymicrogyria transition reveals a requirement for Gpr56 in syndromes. Ann. Neurol. 58, 680–687 (2005). HSC generation. J. Exp. Med. 212, 93–106 (2015). 93 Parrini, E., Ferrari, A. R., Dorn, T., Walsh, 106 Giera, S. et al. The adhesion G protein-coupled C. A. & Guerrini, R. Bilateral frontoparietal receptor GPR56 is a cell-autonomous regulator polymicrogyria, Lennox-Gastaut syndrome, of oligodendrocyte development. Nat Commun and GPR56 gene mutations. Epilepsia 50, 1344– 6, 6121 (2015). 1353 (2009). 107 Chiesa, Della, M. et al. GPR56 as a novel marker 94 Bahi-Buisson, N. et al. GPR56-related bilateral identifying the CD56dull CD16+ NK cell subset frontoparietal polymicrogyria: further evidence both in blood stream and in inflamed peripheral for an overlap with the cobblestone complex. tissues. Int. Immunol. 22, 91–100 (2010). Brain 133, 3194–3209 (2010). 108 Peng, Y.-M. et al. Specific expression of GPR56 95 Luo, R. et al. A novel GPR56 mutation causes by human cytotoxic lymphocytes. J. Leukoc. bilateral frontoparietal polymicrogyria. Pediatr. Biol. 90, 735–740 (2011). Neurol. 45, 49–53 (2011). 109 Sud, N., Sharma, R., Ray, R., Chattopadhyay, 96 Quattrocchi, C. C. et al. Conventional magnetic T. K. & Ralhan, R. Differential expression resonance imaging and diffusion tensor of G-protein coupled receptor 56 in human

28 Introduction

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29 136 Monk, K. R. et al. A G protein-coupled receptor requires the function of gpr126 (lauscher), an is essential for Schwann cells to initiate adhesion class G protein-coupled receptor gene. myelination. Science 325, 1402–1405 (2009). Development 140, 4362–4374 (2013). 137 Waller-Evans, H. et al. The orphan adhesion-GPCR 140 Ni, Y.-Y. et al. Deletion of Gpr128 results in weight GPR126 is required for embryonic development in loss and increased intestinal contraction frequency. the mouse. PLoS ONE 5, e14047 (2010). World J. Gastroenterol. 20, 498–508 (2014). 138 Patra, C. et al. Organ-specific function of 141 Chase, A. et al. TFG, a target of chromosome adhesion G protein-coupled receptor GPR126 is translocations in lymphoma and soft tissue domain-dependent. Proc. Natl. Acad. Sci. U.S.A. tumors, fuses to GPR128 in healthy individuals. 110, 16898–16903 (2013). Haematologica 95, 20–26 (2010). 139 Geng, F.-S. et al. Semicircular canal morphogenesis in the zebrafish inner ear

30