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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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Discussion

This thesis describes research done on the adhesion G protein-coupled receptors (adhesion GPCRs) CD97 and GPR56. Both receptors have been intensively studied in the past, which has resulted in knowledge about their cellular distribution, ligand specificity, signaling capacity, and (patho)physiological activities (see Introduction). This and the availability of a large set of research tools, including expression constructs, monoclonal antibodies, and (not studied here) genetically modified mice, make CD97 and GPR56 useful models for studying structure–function relationships in adhesion GPCRs. Discussed here are wider implications and perspectives in relation to Chapter 2 to 7.

GPS autoproteolysis of adhesion GPCRs Right after cloning of the first adhesion GPCRs, evidence was obtained that some of these receptors are expressed at the cell surface as two non-covalently associated fragments – one N-terminal and one C-terminal – that result from endogenous proteolytic cleavage of a precursor polypeptide1,2. By studying EMR23, Lin et al. demonstrated that the cleavage is self-catalyzed and resembles a mechanism discovered first in cis-proteolytic N-terminal nucleophile hydrolases and confirmed later in an 11TM receptor, called polycystin-14. As cleavage site, aliphatic residues (L↓T/S/C) within a juxtamembranous ~50-amino acid 8 GPCR-proteolytic site (GPS) were identified. Recently, Araç and colleagues showed that the GPS actually is part of much larger, ~320-amino acid GPCR-autoproteolsis inducing (GAIN) domain5. The crystal structure of this GAIN domain has been identified in latrophilin 1 and BAI3. The GAIN domain was shown to be necessary and sufficient for autoproteolysis5. However, not all adhesion GPCRs contain a GPS motif, and also receptors that do not undergo autoproteolysis, such as EMR1, bear a GAIN domain, indicating that this domain has a more general function for the folding of the receptors. A systematic analysis of the cleavage of all human adhesion GPCRs still needs to be performed. Moreover, it needs to be studied whether and how the GPS autoproteolytic reaction is regulated. GPS cleavage happens in the endoplasmic reticulum (ER) immediately after translation of the polypeptide3. Of note, the GAIN–GPS region in adhesion GPCRs and polycystin-1 contains potential N- and O-glycosylation sites, sometimes near the cleavage site. N-glycoslation occurs mainly in the ER and is important for the structure of proteins. Using inhibitors and mutants, we showed in Chapter 2 that GPS autoproteolysis can be modulated by site-specific N-glycosylation at the GAIN–GPS region. Interestingly, a role of N-glycosylation in the autocatalytic cleavage has also been confirmed for polycystin-14. We conclude that cellular functions of adhesion GPCRs can be conditioned by the balance between cleaved and uncleaved receptor molecules that is regulated by the efficiency of site-specific N-glycosylation (Fig. 1). GPS proteolysis has been suggested to affect the maturation, stability, trafficking, and function of adhesion GPCRs6. However, the functional meaning of the proteolytic modification has become a matter of intensive debate recently7. GPS proteolysis is necessary for the maturation and trafficking of some but not all adhesion GPCRs. Moreover, the cleaved N-terminal fragment (NTF) and C-terminal fragment (CTF) can either work together in a

151 AB Cleaved Non‐cleaved

GAIN G1 N‐glycan G2 G3 G4 G5 GPS

Fig. 1. Schematic structure of CD97 (A) Diagram depicting the CD97 receptor with potential N-glycosylation sites in the EGF-like domains and in the GAIN domain, including one at the GPS motif (●). (B) Our results indicate that GAIN domain N-glycosylation sites 4 and 5, but not 1 to 3, are needed for the cleavage of CD97.

heterodimeric receptor complex or independently as separate entities8. Notably, when the NTF is cleaved and shed, the CTF alone drives stronger signaling activity than the full- length receptor in a number of adhesion GPCRs, including GPR56, BAI1, CD97, GPR126, and GPR1339-13. This remarkable observation has raised questions regarding the activation mechanism of adhesion GPCRs (Fig. 2). Does the NTF inhibit receptor activity, and is the NTF released upon ligand binding, possibly facilitated by the natural breakpoint within the GPS motif? Liebscher et al. recently proposed a model according to which the removal of the NTF exposes a short extracellular peptide-sequence to the seven-transmembrane (7TM) domain, which triggers G-protein activation14. A main question that remains to be answered is how ligation of adhesion receptors can result in release of their NTF, which is closely attached to the CTF in the GAIN domain. The Monk group recently showed that GPR126 has domain-dependent functions in Schwann cell development, in which the NTF is essential and sufficient for radial sorting, while the CTF facilitates wrapping14. They postulate that release of the NTF via mechanical forces, induced by polymerization of its ligand laminin-211, enables elevation of cAMP through the remaining CTF. Similarly, the Langenhan group obtained evidence that links function of the adhesion GPCR latrophilin to mechanoreceptivity in sensory neuron populations15. Thus ligation, in combination with mechanical stress, may release the NTF and increase (constitutive) signaling by the CTF (Fig. 2B-a). Responsiveness of adhesion GPCRs to mechanical forces was first described by our group, when we showed that contact of CD97 with its binding partner CD55 under shear stress causes shedding of the NTF16. Notably, release of the NTF was followed almost immediately by disappearance of the CTF, and no evidence for forced CD97 signaling was obtained (Fig. 2B-b). Moreover, soluble CD97 NTF enhanced angiogenesis through interaction with integrins on endothelial cells, indicating that shed NTFs can have autonomous activities (Fig. 2C). Thus, adhesion GPCR fragments seem to function via different mechanisms. Studies with truncated, single-transmembrane CD97 receptors indicated that the CTF is required for receptor function17, but the requirement of the extracellular NTF remained unclear. Chapter 4 of this

152 Discussion

A B C NTF/CTF CTF signaling NTF signaling signaling

a

ON b ON

OFF

Fig. 2. Activation/deactivation of adhesion GPCRs by NTF–CTF interactions (A) Classical model of receptor activation in which ligation (●) of the NTF triggers CTF signaling. (B) Alternatively, NTF shedding resulting from ligand binding in combination with mechanical stress or from secondary cleavage may (a) initiate or (b) terminate receptor signaling. (C) Shed NTFs might consequently induce signals at neighboring cells or at more distant locations. Figure adapted from ref. 1 8 thesis shows that the CD97-associated functions in cell adhesion and migration require the NTF, which cannot be lost. Our data strongly indicate that cooperation with the NTF is necessary for signaling transduction by the CD97 CTF (Fig.2A). The studies of cleavage-deficient mutants in Chapter 3–5 suggest that GPS autoproteolysis plays a critical role in the function of adhesion GPCRs. We propose that functional adhesion GPCRs require proper GPS autoproteolysis supported by post-translational modifications, including sites-specific N-glycosylation.

Functions of CD97 in tumorigenesis Robust CD97 expression is found in many types of tumors18-24 and often correlates with the clinical stage25. CD97 plays multiple roles in tumor progression by affecting migration/invasion17,19,22,26 and angiogenesis26-28 as well as by modulating signaling of the lysophosphatidic acid receptor 1 (LPAR1)28. In Chapter 3 we demonstrated that forced CD97 expression in HT1080 cells enhances the expression of N-cadherin and its intracellular adaptor proteins (β-catenin, α-catenin), which in turn promotes Ca2+-dependent homotypic cell–cell aggregation. Forced expression of CD97 in mouse enterocytes increased the structural integrity of E-cadherin-mediated adherens junctions via enhanced β-catenin, α-catenin, and p120-catenin. As a result, CD97- transgenic mice are more resistant to dextran sodium sulfate-induced colitis29. N-cadherin is broadly expressed in cancer cells and can facilitate survival/apoptosis, migration/ invasion, and epithelial–mesenchymal transition30. β-catenin works as an oncogene and

153 regulates genomic transcription to control neoplastic transformation, progression, survival, and relapse31. Our findings indicate that CD97 enhances the expression of N-cadherin and β-catenin to possibly modulate tumorigenesis. Previous studies revealed that CD97 plays a role in tumor migration/invasion17,19,22,26. In addition, ectopic CD97 expression enhanced cell migration and proteolytic matrix metalloproteinases (MMPs)26-28. Curiously, our current findings in Chapter 4 contradict some of these findings. We demonstrate that enhanced cell adhesion is attributable to upregulated integrins, whereas reduced cell migration/invasion resulted in reduced MT1-MMP and MMP-2 activities, due to induced tissue inhibitor of MMP (TIMP)-2 secretion. The CD97-mediated functions were consistent in cells expressing two different isoforms and were reversed by gene knockdown. In fact, diverse and sometimes contradictory tumorigenic effects of CD97 have been reported before. For instance, it was shown that the CD97(EGF125) isoform induced but the CD97(EGF1-5) isoform suppressed tumor cell invasion28. These variable roles of CD97 in tumorigenesis might be related to several factors, including cell-type specificity, levels of expression, post-translational modifications of CD97, binding partner interactions, and heterodimerization with other surface proteins. Chapter 5 is the first study illustrating a role of an adhesion GPCR in the regulation of apoptosis in tumor cells. Previous studies already suggested that cell survival of non-malignant cells can be regulated by adhesion GPCRs, including cadherin EGF LAG seven-pass G-type receptors (CELSRs)32, brain angiogenesis inhibitor (BAI)133, and GPR5625. We demonstrated that ectopic CD97 expression inhibits both intrinsic and extrinsic apoptosis. Apoptosis is a multistep process tightly controlled by anti- and pro-apoptotic molecules. We applied two independent systems to overexpress CD97 in fibrosarcoma HT1080 cells and quantified several apoptosis-regulating molecules, both at the mRNA and protein level; yet, our experiments failed to disclose a central mechanism by which CD97 protects cells from apoptosis. The work described in this thesis increases our knowledge about the cellular functions of CD97 in tumorigenesis (Fig. 3).

BFPP-associated GPR56 mutations link to multiple molecular mechanisms Bilateral frontoparietal polymicrogyria (BFPP) is a congenital cortex malformation of the brain that has been linked to mutations in the GPR56 gene34. More than 25 disease-associated GPR56 mutations have been found in recent years34-40, but how these mutations affect receptor functions remained poorly understood. Previous reports and our work described in Chapter 6 indicate that disease-associated mutations affect conformational stability and cause defective protein trafficking, both reducing receptor surface expression41,42. Some mutations failed to shed the GPR56 NTF. Although BFPP-associated mutations mainly reduced the surface GPR56 expression, some mutations had no significant effect, indicating that besides reduced surface expression probably other mechanisms can lead to BFPP. Distribution of the NTF and CTF of GPR56 to different membrane raft subdomains

154 Discussion

CD97 Migration/Invasion ↓ (↑) Aggregation ↑ TIMP‐2 ↑ N‐cadherin ↑ Adhesion ↑ Integrin ↑ MMP‐2 MT1‐MMP

α‐catenin ↑ ‐catenin ↑

p120‐catenin ↑ Apoptosis ↓ Caspase cascade ↓

Apoptotic proteins

Fig. 3. CD97 regulates various cellular functions in tumor cells CD97 induces the expression of N-cadherin and its adapter proteins to mediate cell–cell aggregation. Furthermore, CD97 controls the balance between anti- and pro-apoptotic molecules to inhibit caspase-dependent apoptosis. CD97 enhances cell adhesion by upregulating integrins and reduces cell migration/invasion by inhibiting MT1-MMP and MMP-2 activities, due to induced TIMP-2 secretion. Notably, other studies showed a reduction of cell migration/invasion by CD97. These various activities depend on both the NTF and the CTF of CD97. 8 suggests that the two fragments are not always tightly associated on the plasma membrane. Lipid rafts composed of sterols, sphingolipids, and protein are functional platforms for intracellular signaling43. Localization of the CTF in distinct raft regions may play a role in GPR56 signaling. The CTF of disease-associated mutations in the extracellular loops of 7TM domain showed large aggregation and localization in non-raft regions, suggesting impairment of GPR56 function by interfering with intracellular signaling and/or the stability of the CTF. We concluded that BFPP caused by disease-associated GPR56 mutants can result from (the disturbance of) multiple molecular mechanisms: protein trafficking, expression, GPS proteolytic cleavage, NTF shedding, ligand interaction, and differential membrane distribution of receptor fragments. Due to the strong BFPP phenotype, disease-associated GPR56 mutants can help to clarify structure–function relationships and display amino acids that are critical folding and efficient expression of adhesion GPCRs. These amino acids may serve as targets for pharmacological intervention to either increase or inhibit adhesion GPCR signaling.

GPR56 in human cytotoxic lymphocytes CD56dim NK and CD27–CD45RA+ CD8+ T cells are highly reactive cytotoxic lymphocytes that provide protection against virus-infected and transformed cells. Because of their potent killing capacity, these cells need to be tightly controlled by inhibitory receptors in order to prevent autoimmunity. We previously demonstrated that GPR56 is expressed by human cytotoxic lymphocytes but not any other mature human immune cells44. Previous studies

155 in other cell types have implicated roles of GPR56 in generation and maintenance of the hematopoietic stem cell pool, brain development, male fertility, muscle hypertrophy, and melanoma tumorigenesis25,34,45-51. Little was known, however, about the function of GPR56 in human cytotoxic lymphocytes. Moreover, as adhesion GPCR, GPR56 does not belong to a protein family commonly linked to NK-cell regulation, such as immunoglobulin-like receptors and C-type lectins52,53. In Chapter 7, we describe GPR56 as a novel differentiation marker and inhibitory receptor expressed on human CD56dim NK cells. NK cells from two BFPP siblings with the R565W mutation, which did not express any GPR56, developed normally, but killed K562 target cells more efficiently, accompanied by enhanced degranulation and cytokine secretion. Vice versa, NK-92 overexpressing GPR56 cells contained less granzyme B and TNF transcripts at resting state and produced less TNF and IFNγ protein upon PMA stimulation and target cell exposure. Little et al. firstly demonstrated the presence of GPR56–CD81 complexes in the tetraspanin web, which is a functional membrane protein scaffold for regulating signal transmission54. Moreover, CD81 has been found to inhibit NK-cell functions when cross- linked by an antibody55,56. GPR56–CD81 complexes on the NK-cell surface were quickly reduced and moved to the joint points with the target cells, while these complexes were dissociated by GPR56 antibody-ligation, which indicates a role in regulating NK-cell activities. We suggest that GPR56 serves as “endogenous” inhibitory receptor in NK cells that by laterally binding CD81 provides an “internal” inhibitory signal. In humans, homolog of Blimp-1 in T cells (Hobit), a transcriptional repressor closely related to B lymphocyte-induced maturation protein-1 (Blimp-1), is expressed in quiescent effector NK and T cells, which closely matches the expression of GPR56 (Vieira Braga et al., submitted for publication). By studying primary NK cells and cell lines, we obtained evidence that Hobit drives the expression of GPR56. Of note, the GPR56 locus contains 17 transcription start sites in humans that enable binding of different transcription factors45,48, contributing to the broad cellular expression of GPR5657. Hobit comprises homologous DNA- binding zinc finger domains, similar to Blimp-158. Of note, the shared consensus binding sequence for Blimp-1/Hobit – G(T/C)GAAAG(T/C)(G/T) (Vieira Braga et al., submitted for publication) – is present several times in the 5’- region of GPR56 (data not shown). We conclude that GPR56 is a transcriptional target of Hobit in human NK and T cells. The ability to downregulate inhibitory pathways enables effector NK and CD8+ T cells to unfold their full functional capacity. GPR56 is downregulated rapidly and completely by PMA through PKC-mediated shedding and internalization. Moreover, inflammatory conditions, created by IL-15 and IL-1859, caused PKC-dependent shedding of GPR56. As discussed above, receptor shedding is a functional feature of adhesion GPCRs, which can enable the juxtamembranous N-terminal end of the CTF to bind to the 7TM region and cause downstream signaling. Previous studies reported that loss of the NTF strongly enhances CTF signaling of GPR56 and other adhesion GPCRs9-13,60. Moreover, lack of the NTF of GPR56 led to massive ubiquitylation of the CTF9. In cytotoxic lymphocytes, the fate and possible activities of the CTF upon activation-mediated release of the NTF remain to be determined. GPR56 ligands

156 Discussion are collagen III in the developing cerebral cortex and muscle cells and transglutaminase 2 in melanoma cells. Yet, we could not confirm these molecules as binding partner of GPR56 on NK cells. Instead, cellular activation, as discussed above, caused release of the NTF.

Signaling molecules including Gαq/11, Gα12/13, PKCα, RhoA, and mTOR have been linked 9,48,50,51,54,61,62 to GPR56 in different cell types . Of interest is the specific association with Gαq/11. 54 The interaction with CD81 helps to promote/stabilize the GPR56–Gαq/11 association . The

GPR56–CD81–Gαq/11 complex is dynamically regulated: ligation by CD81 antibody caused the release of Gαq/11 from the GPR56–CD81 complex, while PMA stimulation dissociated

GPR56 from CD81–Gαq/11, leading to internalization of GPR56. White et al. reported that GPR56 regulates muscle hypertrophy signaling via Gα12/13-initiated mTOR, a serine- threonine kinase that links various metabolic cues48. In NK cells, high levels of IL-15 lead to mTOR activation63. To explore whether GPR56 affects mTOR in NK cells, we tested the activity of the mTORC1 and mTORC2 complex in NK-92 cells overexpressing GPR56. We found that IL-2-starved NK-92–GPR56 cells more robustly induced mTORC1/2 activity than control cells upon subsequent cytokine stimulation (data not shown), indicating that GPR56 may regulate NK-cell metabolism by mTOR signaling. These preliminary data support a model whereby GPR56 in resting cytotoxic lymphocytes inhibits cytotoxicity via a complex with CD81 and Gα , while upon cell activation, shedding of the NTF q/11 8 dissociates with CD81 and further enables interaction with Gα12/13, leading to RhoA and mTOR signaling as well as incudes receptor degradation (Fig. 4).

Resting cells Activating cells

Cytokines, Target cells

GPR56 CD81 GPR56 CD81

γ Gαq/11 β γ Gα12/13 β

p p Degradation ? mTOR RhoA

Cytotoxicity Metabolism Cytotoxicity Migration Migration

Fig. 4. Functional regulation of human NK cells by GPR56 We hypothesize that GPR56, depending on the cell state – resting versus activated – engages different G-protein signaling pathways. At rest, GPR56 forms a complex with CD81 to inhibit NK-cell migration and target cytotoxicity through Gαq/11 signaling. Upon physiological activation, the NTF of GPR56 is removed by an unknown sheddase, causing dissociation of the CTF from CD81. Loss of the NTF may result in the recruitment of Gα12/13, resulting in mTOR and RhoA signaling and enhanced effector functions.

157 Concluding remarks and future perspectives Since their first description in the mid-1990s, biochemical, genetic, functional, and, more recently, also pharmacological studies have disclosed characteristics of adhesion GPCRs. Nevertheless, many questions remain to be answered, and neither the mechanism of receptor activation nor the link between signaling and function is currently well understood. This thesis contributes to understanding the regulation of GPS autoproteolysis as well as the role of CD97 and GPR56 in tumorigenesis and immunity. Autocatalytic cleavage at the GPS motif within the GAIN domain is a hallmark of adhesion GPCRs and enables the characteristic bipartite structure of most members of this receptor family. While recent studies provided evidence for a critical importance of the separate fragments for adhesion GPCR signaling, we found that biological functions of the receptors depend on the joint presence of both fragments. Transgenic modification of the GPS motif in animal models will be instrumental for further exploring the importance of autoproteolysis for the signaling and functioning of adhesion GPCRs. Ultimately, adhesion GPCR research may lead to applications in molecular medicine. According to our findings on structure regulation, site-specific N-glycan on NTF of adhesion GPCRs might be a good candidate for drug design. Therefore CD97 is an attractive pharmacological target due to its strongly association with human cancer. Our studies on disease-associated mutations in BFPP patients disclosed various potential sites for GPR56 targeting, while our work on the role of GPR56 in NK (and CD8+ T) cells suggests that inhibition of the receptor may enhance the capability of the cells to eradicate tumor. On the other side, NK and T cells have been implicated in the pathogenesis of autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. Here, activation of GPR56 may help to inhibit unwanted NK- and T-cell activity.

158 Discussion

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160 Discussion

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