GPCR Autoproteolysis-inducing Research Articles

Unveiling Mechanical Activation: GAIN Domain Unfolding and Dissociation in Adhesion GPCRs.

Adhesion G protein-coupled receptors (aGPCRs) have extracellular regions (ECRs) containing GPCR autoproteolysis-inducing (GAIN) domains. The GAIN domain enables the ECR to self-cleave into N- and C-terminal fragments. However, the impact of force on the GAIN domain's conformation, critical for mechanosensitive aGPCR activation, remains unclear. Our study investigated the mechanical stability of GAIN domains in three aGPCRs (B, G, and L subfamilies) at a loading rate of 1 pN/s. We discovered that forces of a few piconewtons can destabilize the GAIN domains. In autocleaved aGPCRs ADGRG1/GPR56 and ADGRL1/LPHN1, these forces cause the GAIN domain detachment from the membrane-proximal Stachel sequence, preceded by partial unfolding. In noncleavable aGPCR ADGRB3/BAI3 and cleavage-deficient mutant ADGRG1/GPR56-T383G, complex mechanical unfolding of the GAIN domain occurs. Additionally, GAIN domain detachment happens during cell migration. Our findings support the mechanical activation hypothesis of aGPCRs, emphasizing the sensitivity of the GAIN domain structure and detachment to physiological force ranges.

Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.

Adhesion Class GPCRs in GtoPdb v.2023.1

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [10] containing a GPCR proteolysis site (GPS). The N-terminal extracellular region often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [104, 418]. Several receptors have been suggested to function as mechanosensors [320, 288, 396, 38]. Cryo-EM structures of the 7-transmembrane domain of several adhesion GPCRs have been determined recently [292, 21, 403, 212, 300, 302, 431, 293]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [125].

CSIG-27. MODULATION OF GPR133 (ADGRD1) SIGNALING BY ITS INTRACELLULAR INTERACTION PARTNER EXTENDED SYNAPTOTAGMIN 1 (ESYT1)

Abstract GPR133 (ADGRD1), a member of the adhesion GPCR (aGPCR) family, has been recently implicated in the pathogenesis of glioblastoma. Like other aGPCRs, GPR133 is characterized by a large N-terminus, which possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain catalyzing cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane correlates with increased GPR133 signaling, which is mediated by coupling to Gs and increase in cytosolic cAMP. Since GPR133 is absent in normal brain cells and de novo expressed in GBM, it represents a potential target for GBM treatment. However, little is known about the receptor’s protein interactome and its effects on receptor function and signaling. To identify intracellular interactors of GPR133, we used a proximity biotinylation/mass spectrometry approach in HEK293T cells expressing the wild-type or a signaling-incompetent mutant GPR133. We identified Extended Synaptotagmin 1 (ESYT1), a protein responsible for Ca2+-dependent formation of endoplasmic reticulum-plasma membrane bridges, as the strongest interaction partner for both versions of the receptor. Co-immunoprecipitation confirmed robust binding of ESYT1 to GPR133. Knockdown/knockout of ESYT1 increased GPR133 signaling, while overexpression of ESYT1 had the opposite effect. This effect was not mediated by changes in GPR133 expression or plasma membrane trafficking. We are currently performing additional studies to elucidate the mechanism whereby ESYT1 modulates GPR133 signaling. Collectively, these data suggest that GPR133 signaling is modulated through a novel cytosolic, signaling-independent interaction with ESYT1.

GPR125 (ADGRA3) is an autocleavable adhesion GPCR that traffics with Dlg1 to the basolateral membrane and regulates epithelial apicobasal polarity

The adhesion family of G protein–coupled receptors (GPCRs) is defined by an N-terminal large extracellular region that contains various adhesion-related domains and a highly-conserved GPCR-autoproteolysis-inducing (GAIN) domain, the latter of which is located immediately before a canonical seven-transmembrane domain. These receptors are expressed widely and involved in various functions including development, angiogenesis, synapse formation, and tumorigenesis. GPR125 (ADGRA3), an orphan adhesion GPCR, has been shown to modulate planar cell polarity in gastrulating zebrafish, but its biochemical properties and role in mammalian cells have remained largely unknown. Here, we show that human GPR125 likely undergoes cis-autoproteolysis when expressed in canine kidney epithelial MDCK cells and human embryonic kidney HEK293 cells. The cleavage appears to occur at an atypical GPCR proteolysis site within the GAIN domain during an early stage of receptor biosynthesis. The products, i.e., the N-terminal and C-terminal fragments, seem to remain associated after self-proteolysis, as observed in other adhesion GPCRs. Furthermore, in polarized MDCK cells, GPR125 is exclusively recruited to the basolateral domain of the plasma membrane. The recruitment likely requires the C-terminal PDZ-domain–binding motif of GPR125 and its interaction with the cell polarity protein Dlg1. Knockdown of GPR125 as well as that of Dlg1 results in formation of aberrant cysts with multiple lumens in Matrigel 3D culture of MDCK cells. Consistent with the multilumen phenotype, mitotic spindles are incorrectly oriented during cystogenesis in GPR125-KO MDCK cells. Thus, the basolateral protein GPR125, an autocleavable adhesion GPCR, appears to play a crucial role in apicobasal polarization in epithelial cells.

CSIG-07. ACTIVATION OF THE ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 BY ANTIBODIES AGAINST THE N-TERMINUS

Abstract We recently demonstrated that GPR133 (ADGRD1), a member of the adhesion G protein-coupled receptor (aGPCR) family, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We therefore postulate that GPR133 represents a novel target in GBM, which merits development of therapeutics. Like most aGPCRs, GPR133 is characterized by an intracellular C-terminus, 7 plasma membrane-spanning α-helices and a large extracellular N-terminus. The N-terminus possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain that catalyzes cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane increases canonical signaling of GPR133, which is mediated by coupling to Gs and increase in cytosolic cAMP. Toward characterizing the effect of biologics on GPR133 function, we overexpressed wild-type or mutant forms of GPR133 in HEK293T cells and patient-derived GBM cells lines. Treatment of these cells with antibodies specifically targeting the NTF of GPR133 increased receptor activation in a dose-dependent manner. No effects were elicited with an antibody against the receptor’s intracellular C-terminus. Interestingly, cells overexpressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. Following antibody treatment, co-purification of the GPR133 NTF and the N-terminal antibody from the cell culture supernatant indicated the formation of antibody-NTF complexes. Analysis of these complexes suggested that antibody binding stimulated the dissociation of the NTF from the CTF. However, the increased flexibility of the GAIN domain and NTF after cleavage, independently of dissociation, may also endow the receptor with responsiveness to the effects of the antibodies. These data constitute a proof-of-concept paradigm of modulation of GPR133 function with antibodies. This work provides rationale for pursuing development of biologics targeting GPR133 in GBM.

Adhesion Class GPCRs in GtoPdb v.2021.3

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [9] containing a GPCR proteolytic site. The N-terminus often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [101, 403]. Several receptors have been suggested to function as mechanosensors [309, 280, 383, 35]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [122].

Emerging roles of adhesion G protein-coupled receptors.

Adhesion G protein-coupled receptors (aGPCRs) form a sub-group within the GPCR superfamily. Their distinctive structure contains an abnormally large N-terminal, extracellular region with a GPCR autoproteolysis-inducing (GAIN) domain. In most aGPCRs, the GAIN domain constitutively cleaves the receptor into two fragments. This process is often required for aGPCR signalling. Over the last two decades, much research has focussed on aGPCR-ligand interactions, in an attempt to deorphanize the family. Most ligands have been found to bind to regions N-terminal to the GAIN domain. These receptors may bind a variety of ligands, ranging across membrane-bound proteins and extracellular matrix components. Recent advancements have revealed a conserved method of aGPCR activation involving a tethered ligand within the GAIN domain. Evidence for this comes from increased activity in receptor mutants exposing the tethered ligand. As a result, G protein-coupling partners of aGPCRs have been more extensively characterised, making use of their tethered ligand to create constitutively active mutants. This has led to demonstrations of aGPCR function in, for example, neurodevelopment and tumour growth. However, questions remain around the ligands that may bind many aGPCRs, how this binding is translated into changes in the GAIN domain, and the exact mechanism of aGPCR activation following GAIN domain conformational changes. This review aims to examine the current knowledge around aGPCR activation, including ligand binding sites, the mechanism of GAIN domain-mediated receptor activation and how aGPCR transmembrane domains may relate to activation. Other aspects of aGPCR signalling will be touched upon, such as downstream effectors and physiological roles.

Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.

Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding

Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.

Adhesion Class GPCRs (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [8] containing a GPCR proteolytic site. The N-terminus often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [82, 332]. Several receptors have been suggested to function as mechanosensors [254, 234, 315, 32]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [100].

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

Membrane-association of EMR2/ADGRE2-NTF is regulated by site-specific N-glycosylation

The evolutionarily conserved adhesion G protein-coupled receptors (aGPCRs) play critical roles in biological processes as diverse as brain development, cell polarity and innate immune functions. A defining feature of aGPCRs is the GPCR autoproteolysis inducing (GAIN) domain capable of self-catalytic cleavage, resulting in the generation of an extracellular N-terminal fragment (NTF) and a seven-transmembrane C-terminal fragment (CTF) involved in the cellular adhesion and signaling functions, respectively. Interestingly, two different NTF subtypes have previously been identified, namely an NTF that couples non-covalently with the CTF and a membrane-associated NTF that tethers on cell surface independently. The two NTF subtypes are expected to regulate aGPCR signaling via distinct mechanisms however their molecular characteristics are largely unknown. Herein, the membrane-associated NTF of EMR2/ADGRE2 is investigated and found to be modified by differential N-glycosylation. The membrane association of EMR2-NTF occurs in post-ER compartments and site-specific N-glycosylation in the GAIN domain is involved in modulating its membrane-association ability. Finally, a unique amphipathic α-helix in the GAIN domain is identified as a putative membrane anchor of EMR2-NTF. These results provide novel insights into the complex interaction and activation mechanisms of aGPCRs.

Adhesion G Protein-Coupled Receptors as Drug Targets.

The adhesion G protein-coupled receptors (aGPCRs) are an evolutionarily ancient family of receptors that play key roles in many different physiological processes. These receptors are notable for their exceptionally long ectodomains, which span several hundred to several thousand amino acids and contain various adhesion-related domains, as well as a GPCR autoproteolysis-inducing (GAIN) domain. The GAIN domain is conserved throughout almost the entire family and undergoes autoproteolysis to cleave the receptors into two noncovalently-associated protomers. Recent studies have revealed that the signaling activity of aGPCRs is largely determined by changes in the interactions among these protomers. We review recent advances in understanding aGPCR activation mechanisms and discuss the physiological roles and pharmacological properties of aGPCRs, with an eye toward the potential utility of these receptors as drug targets.

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment.

Stalk‐dependent and Stalk‐independent Activity of the Adhesion GPCR GPR56/ADGRG1

The adhesion G protein‐coupled receptors (aGPCRs) comprise an enigmatic family of 33 receptors in humans. A hallmark of the aGPCRs is the presence of a large extracellular N‐terminal (NT) domain that has been shown to inhibit receptor activity. Yet another defining characteristic of virtually all aGPCRs is the presence of a GPCR Autoproteolysis Inducing (GAIN) domain, which is found on the membrane‐proximal end of the NT and controls the autocatalytic proteolysis of the receptor into an NT protomer and seven‐transmembrane (7TM) protomer, which can stay non‐covalently associated for some time. Recently, great progress has been made to shed light upon a more detailed view of how exactly the NT regions inhibit aGPCR‐mediated signaling. Notably, two separate groups have espoused a “cryptic agonist” model of aGPCR activation wherein GAIN domain cleavage unveils a new NT stalk which serves as the receptor's agonist. In this model, the stalk is a requisite agonist for aGPCR activity and deletion of the stalk result in a signaling‐incompetent receptor. In the work presented here, we show that the stalk of the aGPCR GPR56/ADGRG1 (“G1”) is indeed necessary for activation of the certain downstream readouts (such as activation of SRF luciferase) but dispensable for others (such as the shedding of membrane‐bound TGFalpha and activation of NFAT luciferase). Moreover, a stalkless version of G1 also displays comparable activity to the highly‐active, GAIN‐cleavage‐mimetic form of G1 (G1‐deltaNT) in several measures of activity including beta‐arrestin recruitment, receptor ubiquitination, and association with Galpha13. These lines of evidence suggest that G1 is capable of both stalk‐dependent and stalk‐independent modes of signaling. Based upon our results, we propose a more nuanced model of aGPCR activation in which the NT inhibits activity of the 7TM region in two distinct ways: i) antagonizing stalk‐dependent signaling by masking the cryptic stalk peptide, and ii) directly antagonizing the inherent stalk‐independent activity of the 7TM protomer.Support or Funding InformationThis work was supported by NIH R01 award R01‐NS072394.

Control of Adhesion GPCR Function Through Proteolytic Processing.

Proteolytic processing events in adhesion GPCRs. aGPCRs can undergo multiple autoproteolytic (red asterisks) and proteolytic processing events by exogenous proteases (yellow asterisks) that may be involved in signaling events of the receptors. Proteolytic processing is an unusual property of adhesion family G protein-coupled receptors (aGPCRs) that was observed upon their cloning and biochemical characterization.Ever since, much effort has been dedicated to delineate the mechanisms and requirements for cleavage events in the control of aGPCR function. Most notably, all aGPCRs possess a juxtamembrane protein fold, the GPCR autoproteolysis-inducing (GAIN) domain, which operates as an autoprotease for many aGPCR homologs investigated thus far. Analysis of its autoproteolytic reaction, the consequences for receptor fate and function, and the allocation of physiological effects to this peculiar feature of aGPCRs has occupied the experimental agenda of the aGPCR field and shaped our current understanding of the signaling properties and cell biological effects of aGPCRs. Interestingly, individual aGPCRs may undergo additional proteolytic steps, one of them resulting in shedding of the entire ectodomain that is secreted and can function independently. Here, we summarize the current state of knowledge on GAIN domain-mediated and GAIN domain-independent aGPCR cleavage events and their significance for the pharmacological and cellular actions of aGPCRs. Further, we compare and contrast the proteolytic profile of aGPCRs with known signaling routes that are governed through proteolysis of surface molecules such as the Notch and ephrin pathways.

Classification, Nomenclature, and Structural Aspects of Adhesion GPCRs.

Representation of the nine distinct aGPCR subfamilies and their unique N-terminal domain architecture. The illustration also shows the extracellular structural feature shared by all aGPCRs (except ADGRA1), known as the GPCR autoproteolysis-inducing (GAIN) domain, that mediates autoproteolysis and subsequent attachment of the cleaved NTF and CTF fragments The adhesion family of G protein-coupled receptors (aGPCRs) is unique among all GPCR families with long N-termini and multiple domains that are implicated in cell-cell and cell-matrix interactions. Initially, aGPCRs in the human genome were phylogenetically classified into nine distinct subfamilies based on their 7TM sequence similarity. This phylogenetic grouping of genes into subfamilies was found to be in congruence in closely related mammals and other vertebrates as well. Over the years, aGPCR repertoires have been mapped in many species including model organisms, and, currently, there is a growing interest in exploring the pharmacological aspects of aGPCRs. Nonetheless, the aGPCR nomenclature has been highly diverse because experts in the field have used different names for different family members based on their characteristics (e.g., epidermal growth factor-seven-span transmembrane (EGF-TM7)), but without harmonization with regard to nomenclature efforts. In order to facilitate naming of orthologs and other genetic variants in different species in the future, the Adhesion-GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposed a unified nomenclature for aGPCRs. Here, we review the classification and the most recent/current nomenclature of aGPCRs and as well discuss the structural topology of the extracellular domain (ECD)/N-terminal fragment (NTF) that is comparable with this 7TM subfamily classification. Of note, we systematically describe the structural domains in the ECD of aGPCR subfamilies and highlight their role in aGPCR-protein interactions.

Versatile Signaling Activity of Adhesion GPCRs.

The adhesion G protein-coupled receptors (aGPCRs) are a family of 33 receptors in humans that are widely expressed in various tissues and involved in many diverse biological processes. These receptors possess extremely large N-termini (NT) containing a variety of adhesion domains. A distinguishing feature of these receptors is the presence within the NT of a highly conserved GPCR autoproteolysis-inducing (GAIN) domain, which mediates autoproteolysis of the receptors into N-terminal and C-terminal fragments that stay non-covalently associated. The downstream signaling pathways and G protein-coupling preferences of many aGPCRs have recently been elucidated, and putative endogenous ligands for some aGPCRs have also been discovered and characterized in recent years. A pivotal observation for aGPCRs has been that deletion or removal of the NT up the point of GAIN cleavage results in constitutive receptor activation. For at least some aGPCRs, this activation is dependent on the unmasking of specific agonistic peptide sequences within the N-terminal stalk region (i.e., the region between the site of GAIN domain cleavage and the first transmembrane domain). However, the specific peptide sequences involved and the overall importance of the stalk region for activation can vary greatly from receptor to receptor. An emerging theme of work in this area is that aGPCRs are capable of versatile signaling activity that may be fine-tuned to suit the specific physiological roles played by the various members of this family.

Introduction: History of the Adhesion GPCR Field.

Development of the aGPCR scientific fieldbased on PubMed-listed research articles and selected key findings Since the discovery of adhesion G-protein-coupled receptors (aGPCRs) 20 years ago, reverse genetics approaches have dominated the elucidation of their function and work mechanisms. Seminal findings in this field comprise the description of aGPCRs as seven-transmembrane (7TM) molecules with an extended extracellular region, the identification of matricellular ligands that bind to distinct protein folds at the N-terminus, the clarification of an autoproteolytic cleavage event at a juxtamembranous GPCR proteolysis site (GPS), the elucidation of the crystal structure of the GPCR autoproteolysis-inducing (GAIN) domain that embeds the GPS and connects the receptor fragments, the demonstration that a short N-terminal sequence of the seven-transmembrane (7TM) region can serve as a tethered agonist, and, recently, the notification that aGPCRs can serve as mechanosensors. We here discuss how these discoveries have moved forward aGPCR research and, finally, linked the field to the GPCR field. We argue that crucial questions remain to be addressed before we can fully appreciate the biological nature of these fascinating receptors.