UvA-DARE (Digital Academic Repository) Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function Hsiao, C.-C. Publication date 2015 Document Version Final published version Link to publication Citation for published version (APA): Hsiao, C-C. (2015). Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) 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 proteins (G proteins); therefore, they are often called G protein-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, Rhodopsin, Adhesion, Frizzled/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-arrestin, 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-arrestins, 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 (latrophilins), ADGRA, ADGRC (CELSRs), ADGRD, ADGRG, and ADGRV are evolutionary old, while the subfamilies ADGRE (EGF-TM7), ADGRF, and