Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Adhesion GPCRs

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_526

Synonyms

Historical Background

Among the superfamily of G-protein-coupled receptors, the adhesion GPCRs form the second largest family with 33 members in humans (for review, see Bjarnadottir et al. 2007). They are also the most diverse and complex GPCR family, often encoded by very large genes with numerous introns, and comprising highly diverse and variable N-terminal extracellular regions. Despite a remarkable structural diversity and low sequence homology, hydropathy analysis and biochemical data suggest that the adhesion GPCRs share the structural hallmark of all GPCRs – a heptahelical conformation with extracellular N-terminus and intracellular C-terminus. Approximately 150 distinct orthologues have been identified in the animal kingdom to date. The name adhesion GPCRs emphasizes the presence of multiple motifs in their long extracellular N-termini predicting adhesive properties, such as cadherin-, laminin-, lectin-, epidermal growth factor-, olfactomedin-, immunoglobulin-, calcium exchanger β-, thrombospondin-, and mucin-like domains (see Fig. 1).
Adhesion GPCRs, Fig. 1

A schematic presentation of the functional domains found in the N-termini of human adhesion GPCRs (Reproduced with permission from Bjarnadottir et al. 2007). GPS GPCR proteolysis site, HBD hormone binding domain, TSP1 thrombospondin type 1 repeat, PTX Pentraxin doamain, EGF_CA calcium-binding EGF-like domain, OLF Olfactomedin-like domain, GBL galactose binding lectin domain, CA Cadherin-like (Calcium-dependent adhesion-like) domains, LamG Laminin G domain, EGF_Lam EGF-like, fibronectin type III and laminin G domains, LRR leucine-rich repeats, Ig Immunoglobulin-like domains, SEA Sea-urchin sperm protein, enterokinase and agrin module, Calx-beta motif in Na-Ca exchangers and integrin-beta4, CUB Domain first found in complement C1r, C1s, uEGF, and bone morphogenetic protein. Subdivision into different groups or clans (Roman numerals I–VII) was based on the sequence similarity within the heptahelical regions and intervening loops. According to a more recent classification, Group III can be further divided into two clusters (Lagerstrom and Schioth 2008). The first cluster contains LEC1–3 and ETL receptors, whereas the second cluster includes EMR1–4 and CD97 receptors

The Epidermal growth factor Module-containing Receptor EMR1, its mouse homologue F4/80, and CD97, all members of the Epidermal Growth Factor (EGF)-TM7 receptor subfamily, were the first-described adhesion GPCRs (for review, see McKnight and Gordon 1996, 1998). Derived from a single precursor polypeptide, they are expressed at the surface of leukocytes in two parts, (1) a large extracellular domain with variable numbers of modular EGF-like domains and (2) a membrane-spanning domain consisting of seven hydrophobic stretches connected by three intracellular and three extracellular loops. Both parts are associated via a highly glycosylated mucin-like “stalk” which serves as a spacer. The unusual structure of their extracellular regions indicated a function related to cell adhesion and leukocyte migration. Indeed, the ability to bind to extracellular matrix molecules and cellular ligands has been experimentally demonstrated for several members of the EGF-TM7 multigene family (see below).

The subsequent discovery of an increasing number of GPCR-related molecules with a similar domain configuration led to the proposal to reclassify this group of heptahelical membrane proteins as a novel class of receptors (for review, see Stacey et al. 2000). Following the example of the EGF-TM7 receptors, the entire class was termed the LNB-TM7 receptors, where LN stands for their large and complex N-terminal extracellular regions, and B indicates a significant amino acid sequence similarity to G-protein-coupled receptor family B, also named the Secretin GPCRs (following the GRAFS classification system, Fredriksson et al. 2003). Progress in molecular biology and bioinformatics techniques led to the sequencing and assembly of whole genomes, and allowed the identification of the full repertoire of human GPCRs. This enabled a phylogenetic analysis based on the complete sequences of the TM7 regions, which demonstrated that the adhesion GPCRs did indeed form a distinct receptor family (Fredriksson et al. 2003). Based on the sequence similarity within their heptahelical regions and intervening loops, the human adhesion GPCRs were further subdivided into different groups or clans (Fig. 1; for review, see Bjarnadottir et al. 2007; Lagerström and Schiöth 2008). Interestingly, this group structure at the same time reflected a functional classification based on the various adhesion motifs present in the N-termini (Fig. 1). Also, receptors within the same phylogenetic clusters showed similar expression profiles in human tissues.

More recently, Schiöth and coworkers suggested that the adhesion GPCRs were an ancient receptor class, and the Secretin GPCRs descended from a subgroup during metazoan evolution (Nordström et al. 2009). Secretin GPCRs bind polypeptide hormones at a hormone-binding domain (HBD) in their N-termini, and, in response to ligand binding, signal via G-protein activation, predominantly GαS. Although several adhesion GPCRs contain a HBD motif in their N-termini as well (compare Fig. 1), no hormone ligands have been identified for this receptor family, and there is only scant evidence of receptor activation and G-protein coupling (see below). Adhesive interactions have been verified for some family members (see below) while the functional role of a majority of adhesion GPCRs is still poorly understood. Thus, their description as LNB-TM7 receptors may still be more correct, although the name adhesion GPCRs now prevails in the literature.

Posttranslational Processing

The adhesion GPCRs are chimeras, composed of a hydrophilic extracellular adhesion part and a hydrophobic membrane-spanning signaling part. This chimeric configuration already suggested a dual function, combining interactions with the surrounding extracellular matrix and/or cellular counter receptors with coupling to the intracellular signaling machinery. Different from other GPCRs, however, the “mature” adhesion GPCRs almost invariably consist of two separate subunits which appear to be a defining feature of this receptor class. The two subunits are derived from cleavage at the so-called GPCR proteolytic site (GPS), a characteristic Cys-box-containing motif located in the ectodomain adjacent to the first transmembrane domain (Krasnoperov et al. 1997, 2002; Chang et al. 2003; Lin et al. 2004; Figs. 1 and 2). This site is also involved in the cleavage of other membrane-spanning proteins, but is not found in any other GPCR family in the human and mouse genomes.
Adhesion GPCRs, Fig. 2

Protein sequence alignment of the juxtamembrane region of adhesion GPCR family members (modified from Abe et al. 2002). Conserved amino acid residues of the GPCR proteolysis site (GPS) are highlighted. The arrow points to the hypothetical cleavage site within the GPS domain. Amino acid sequences obtained from the NCBI database were aligned with the ClustalW program followed by manual adjustment. 7 TM – adhesion GPCRs, h human, m mouse, r rat, 1 TM – suREJ1 sea urchin Receptor for Egg Jelly 1, 11 TM – suREJ3 sea urchin Receptor for Egg Jelly 3, TM1 first amino acid of transmembrane domain 1

The GPS is one of the few conserved motifs present in all the N-termini of adhesion GPCRs, except GPR123 (see Fig. 1). In addition to this site, most adhesion GPCRs share a second homologous stretch in their extracellular domains, a serine-, threonine-, and proline-rich (STP) “stalk,” typical of a highly O-glycosylated protein domain. Conservation of these motifs in nearly all adhesion GPCRs suggests an important role. The first functional hint of the GPS motif was revealed when intracellular proteolytic processing of cell surface marker CD97 (cluster of differentiation) was demonstrated (Gray et al. 1996). Translated as a single polypeptide, CD97 cleavage results in the formation of the two subunits. Cloning and Western blot analysis of the lectin-like domain-containing calcium-independent receptor of α-latrotoxin (Krasnoperov et al. 1997; Lelianova et al. 1997), GPR116/Ig-Hepta (Abe et al. 2002) and the GPR64/HE6 receptor (Obermann et al. 2003) has revealed additional “split” receptors. The exact cleavage site was determined for CIRL/Latrophilin/lectomedin receptor 1 (Krasnoperov et al. 2002) and also for the EGF-TM7 receptors (Chang et al. 2003). It is located at homologous sites in the C-terminal part of the GPS domain (see Fig. 2).

The GPS motif is necessary, but not sufficient for cleavage. Rather, cleavage also requires the presence of the “stalk” next to the membrane (Chang et al. 2003; Hsiao et al. 2009). As an example, the ectosubunit of the GPR64/HE6 receptor, which is predominantly expressed in the microvilli of the epididymal duct epithelium (Obermann et al. 2003; Kirchhoff et al. 2008), is characterized by the presence of 20 potential N-glycosylation (Asn-Xaa-Ser/Thr) sites and more than 100 potentially O-glycosylated Ser and Thr residues, largely located within the STP region, and most probably forming a hydrophilic mucin-like “stalk” (Obermann et al. 2003). From the large number of glycosylation sites and helix-breaking proline residues, the ectosubunit was predicted to be highly charged and extend from the cell surface like a rod. Similar structural predictions have been made for other adhesion GPCRs, including the EGF-TM7 receptors and the latrophilins (LPHN; synonyms CL/CIRL/Lph). More recently, glycosylation at specific sites of the EGF-TM7 “stalk” has been implicated in cleavage at the GPS (Hsiao et al. 2009).

Proteolytic cleavage is a well-known regulatory mechanism for receptor activation. For example, the G-protein-coupled protease-activated receptors (PARs) signal in response to N-terminal cleavage by extracellular proteases. The new N-terminus then acts as a tethered ligand and binds intramolecularly to the receptor to trigger transmembrane signaling. The mechanism and role of GPS-mediated cleavage seems to be different. Experiments on EMR2, member of the EGF-TM7 family, suggested that it occurs intracellularly in the endoplasmic reticulum (Lin et al. 2004). Whether it could trigger or modulate any signaling events, in analogy to the PAR example, remained unknown. A role in signaling, however, was proposed for GPS cleavage of the Cadherin, EGF LAG seven-pass G-type receptor (CELSR) family members. The extracellular subunits of CELSR2 and CELSR3, including their Cadherin repeats, were proposed to act as a ligand of the seven-transmembrane-spanning part, stimulating calcium release and regulating neurite growth (Shima et al. 2007).

Different from PAR activation, GPS-mediated cleavage is mediated by an autocatalytic reaction rather than by protease activity (Lin et al. 2004). The mechanism is similar to that of N-terminal nucleophile hydrolases, involving the generation and hydrolysis of a (thio)-ester intermediate. Yet, GPS-mediated cleavage appears to be a regulated process, requiring additional modifications like phosphorylation (Kaur et al. 2005) and glycosylation (Hsiao et al. 2009). Endogenous cleavage of the precursor protein may be incomplete; however, full-size, non-cleaved molecules are not normally found in tissues and may not reach the cell surface. Indeed, efficient latrophilin/CIRL receptor trafficking to the cell surface requires proper GPS cleavage (Volynski et al. 2004). Furthermore, deficiency in GPS cleavage caused by mutations has been linked to various human genetic disorders and diseases, including bilateral frontoparietal polymicrogyria (BFPP), and autosomal dominant polycystic kidney disease (ADPKD), emphasizing the critical role of this modification (for review, see Yona et al. 2008a).

Surprisingly, GPS-mediated cleavage of adhesion GPCRs does not yield much soluble ectoprotein; rather, both subunits remain frequently tethered at the cell membrane. Observations from different subgroups showed that the extracellular subunits behave as membrane proteins despite a lack of transmembrane regions (Gray et al. 1996; Krasnoperov et al. 1997, 2002; Obermann et al. 2003). Initially, this was thought to be mediated by a non-covalent interaction with the TM7 domain subunit. However, in the case of latrophilin/CIRL and the EGF-TM7 receptors, the two subunits are delivered to different plasma membrane domains, are recycled separately, and can be patched independently by specific antibodies (Volynski et al. 2004; Davies et al. 2007; Silva et al. 2009a). Apparently, both subunits behave as independent membrane proteins, the ectosubunit being anchored in the plasma membrane by an unknown mechanism. Similarly, both GPR64/HE6 subunits were individually solubilized from different parts of the epididymis (Obermann et al. 2003; Davies et al. 2004). In membrane fractions prepared from the proximal part of this organ, the amount of extractable ectosubunit by far exceeded that of the endosubunit. This peculiar observation was in apparent discrepancy with the expected 1:1 stoichiometry of subunits. While the significance of this observation remains unclear, it may reflect different solubilization characteristics of the two subunits, rather than different locations. Despite their independence, however, subunits of latrophilin/CIRL and the EGF-TM7 receptors can reassemble in the plasma membrane to form heterodimers (Davies et al. 2007; Silva et al. 2009a). This observation has led to the concept of a “split personality” receptor (Volynski et al. 2004; see Fig. 3 and below) which may also be applicable to other adhesion GPCRs.
Adhesion GPCRs, Fig. 3

Proposed scheme of latrophilin/CIRL (LPH) processing, activation, and internalization (modified from Volynski et al. 2004). LPH is cleaved intracellularly in the ER. Proteolysis is required for the delivery of the mature, “split” receptor protein to the cell surface. Further posttranslational processing in the Golgi complex (GC) involves glycosylation and may endow the N-terminal fragment with an own membrane anchor; however, the structure of this anchor is currently unknown. On the cell surface, the two subunits behave as independent membrane proteins. Agonist binding to the ectosubunit promotes its association with the endosubunit and or receptor dimerization, presumably leading to the activation of endosubunit-mediated cell signaling pathways. Internalization of subunits may be independent processes.

Nevertheless, there are also reports of “shedding” of the extracellular subunit. Increased expression of CD97 at sites of inflammation, for example, is accompanied by detectable levels of soluble extracellular subunit (Gray et al. 1996). The mechanism of this release is not known but might include augmented matrix metalloproteinase activity in a pathological situation. The Brain Angiogenesis Inhibitor 1 (BAI1), on the other hand, seems to release its extracellular subunit in the normal brain. It was inferred that the secreted fragment, named Vasculostatin for its antiangiogenic function, resulted from BAI1 cleavage at the GPS (Kaur et al. 2005). However, the precise cleavage site(s) was not determined. It could be that such “shedding” requires cleavage of the BAI1 molecule at multiple sites (compare Koh et al. 2004) as was shown for other adhesion GPCRs (see below). In summary, GPS cleavage which occurs exactly between the two structurally and functionally different domains is an intrinsic posttranslational modification of many, if not most, adhesion GPCRs. For the family members studied, it occurred largely autocatalytically inside the cell, probably at the endoplasmic reticulum (ER). It is a prerequisite for receptor trafficking and functional expression at the cell surface; however, it may not be a common step in signaling.

Soluble fragments may also be generated by alternative mRNA splicing, providing a mechanism for the generation of soluble ligand or receptor desensitization (for review, see Yona et al. 2008a). For a number of adhesion GPCRs, there is evidence for additional proteolytic cleavage events which might lead to the release of N-terminal fragments. CIRL/Latrophilin can be cleaved in vivo by an unknown protease in the remaining short N-terminal stretch of the seven-transmembrane-spanning endosubunit to yield a small 15 amino acid residue oligopeptide (Krasnoperov et al. 2009). While the majority of receptor molecules remained membrane-bound after cleavage at the GPS (see above), this additional cleavage resulted in the formation of soluble subunits. It was assumed that the soluble fragments contained the non-covalently bound 15-mer at their C-terminal ends (Krasnoperov et al. 2009).

Results by Koh et al. (2004) indicated that the extracellular region of brain-specific angiogenesis inhibitor 1 (BAI1) was cleaved at three sites, and that the BAI1-thrombospondin type 1 repeat fragment was the core extracellular fragment for BAI1’s antiproliferative activity. A specifically complex processing was described for Ig-Hepta/GPR116 (Fukuzawa and Hirose 2006), an adhesion GPCR of unknown function which is abundantly expressed in lung and kidney (Abe et al. 2002). These authors further suggested involvement of some fragment(s) in cellular signaling (Fukuzawa and Hirose 2006). GPS-independent cleavage was also described for GPR126/DREG/VIGR (Moriguchi et al. 2004), an adhesion GPCR which was recently shown to be essential for myelination in Schwann cells (Monk et al. 2009). Besides cleavage at the GPS, the protein was further cleaved in the middle of the extracellular domain, generating a soluble fragment containing the CUB (for complement C1r/C1s, Uegf, Bmp1) and pentraxin (PTX) domains (Moriguchi et al. 2004). This processing step was inhibited by an inhibitor of furin but not of matrix metalloproteinases. It was speculated that the subfragment could play a role as a secreted ligand (Moriguchi et al. 2004).

It is unknown whether non-GPS cleavage is a common phenomenon of adhesion GPCRs, and its functional significance remains unclear. Members of other GPCR families, including the V2 receptor and endothelin B receptor, undergo a ligand-induced proteolysis to produce peptides with possible bioactivity. Similarly, the liberated fragments of adhesion GPCRs might be a physiological ligand; alternatively, the cleavage might unmask a hidden ligand binding site, or may activate the receptor (for review, see Yona et al. 2008a). For latrophilin, it was suggested that the non-GPS cleavage(s) served to release defective receptor protein from the cell membrane and/or, in conjunction with GPS cleavage, control cell surface expression (Krasnoperov et al. 2009). Finally, regulated intramembrane proteolysis of membrane receptors produces C-terminal fragments that function inside the cell, even in the nucleus. For most adhesion GPCRs, however, it is as yet unclear whether such C-terminal fragments are generated, probably due to the fact, that the appropriate assays have not been performed.

Ligand Binding

In the absence of endogeneous ligands, a first key concept of ligand binding and activation came from studies involving an exogenous agonist, α-latrotoxin. It is a component of the black widow spider venom, and an activating ligand (=agonist) of CIRL/latrophilin (Krasnoperov et al. 1999). α-latrotoxin binding occurs as two sequential steps: (1) the toxin interacts with the ectodomain with medium affinity followed by (2) an interaction with either the first transmembrane domain, with the membrane lipids, or with both. As a result α-latrotoxin penetrates into the lipid bilayer. The second step increases the affinity of the interaction and requires a longer time. The first site of agonist binding encompassed an area comprising the HBD, stalk, and GPS domain while the N-terminal rhamnose lectin-like (RBL) and olfactomedin-like motifs were not required (Krasnoperov et al. 2002). Also, the extracellular loops of the heptahelical domain were apparently not necessary to stabilize the complex with α-latrotoxin (Krasnoperov et al. 1999). It should, however, be kept in mind that this mechanism of interaction may be completely different from the interaction with a putative endogenous CIRL ligand. The recent identification of a latrophilin-associated synaptic surface protein (“Lasso”) may be the first step to elucidate the interaction with a physiological ligand (Silva et al. 2009b).

The α-latrotoxin receptor is highly conserved among metazoa; in Caenorhabditis elegans the homologous lat-1 is required to coordinately orient cells along a two-dimensional plane lying orthogonal to the axis of apical-basal polarity. Interestingly, different from its mammalian homologue, the extracellular RBL domain of lat-1 was absolutely required for receptor function; constructs lacking the RBL domain but retaining the HBD, GPS, and TM7 domains did not show any biological activity in the C. elegans model (Langenhan et al. 2009). This result is consistent with an essential role of the RBL domain in ligand binding; however, results concerning the structure of this domain argued strongly against a carbohydrate ligand for lat-1.

Concerning the binding of endogenous ligands, members of the EGF-TM7 clan are probably the best studied group. Their ligands are large cell-surface proteins and/or components of the extracellular matrix (see below), justifying the name adhesion GPCRs. Still, even for this subfamily it remains unclear whether ligand binding of ectosubunits leads to the activation of endosubunit-mediated cell signaling. CD97, the prototypic EGF-TM7 receptor on leukocytes, was the first receptor for which a cellular ligand, CD55 (also termed decay-accelerating factor, DAF), was demonstrated (Hamann et al. 1996). CD55/DAF is a GPI-anchored molecule expressed by all blood cells and cells in contact with blood and tissue fluid. CD97 binds CD55/DAF via the N-terminal EGF-like domain region (Hamann et al. 1996, 1998; Lin et al. 2001). This was not unexpected as the EGF-like short consensus repeats are the most characteristic structural elements in the extracellular domain, and are common modules used in cell adhesion and chemotaxis. As a result of alternative mRNA splicing, CD97 exists as three major isoforms that contain between three and five EGF-like domains (compare Fig. 4).
Adhesion GPCRs, Fig. 4

Cartoon representation of CD97 interacting with its cellular ligands (Reproduced with permission from: Hamann J, Veninga H, de Groot DM et al. CD97 in leukocyte trafficking. In: Yona S, Stacey M, eds. Adhesion-GPCRs: structure to function. Austin/New York: Landes Bioscience/Springer Science + Business Media, 2010; epub ahead of print http://www.landesbioscience.com/curie/chapter/4547/.). (a) At the cell surface, CD97 is expressed as a non-covalently associated heterodimer consisting of an extracellular chain and a membrane-spanning chain. The two chains result from autocatalytic processing of a single CD97 propeptide. Alternative splicing generates isoforms with 3, 4, or 5 EGF domains. Shown here are the smallest and the largest isoform. While EGF domains 1 and 2 interact with CD55, EGF domain 4, which only is present in the largest isoform, binds chondroitin sulfate. Integrins bind a RGD motif in the “stalk” region of human CD97. (b) Mouse CD97 has a similar structure but the maximum number of EGF domains is only 4. Shown here is the middle isoform. In the largest isoform of mouse CD97, the EGF domains 2 and 3 are separated by 45 amino acids. (c) Comparison of binding characteristics of human and mouse CD97 isoforms. Depicted is the composition of the EGF domain region, the relative amount of transcripts present in leukocytes and the ligand specificity. In humans, affinity for CD55 correlates inversely with the number of EGF domains. An interaction of EGF domain 3 of mouse CD97 (the counterpart of EGF domain 4 in humans) with chondroitin sulfate still needs to be proven. The binding site of monoclonal antibodies recognizing specific EGF domains in mouse CD97 is indicated

All human CD97 isoforms bind CD55/DAF, albeit with different affinities, the smallest isoform binding the ligand with the highest affinity (Hamann et al. 1996, 1998; Lin et al. 2001). The functional consequences of CD97-CD55 binding have not been fully elucidated, and G-protein-mediated signal transduction has not been demonstrated (see below). The largest isoform of human CD97 also interacts with the glycosaminoglycan chondroitin sulfate B (CS; Stacey et al. 2003; Kwakkenbos et al. 2005; compare Fig. 4). Ligand specificity for CS is shared by EMR2, whose EGF domain region is highly similar to that of CD97, but not by other family members. Indeed, only marginal sequence differences in the EGF-like domains result in dramatic differences in their affinity toward the ligand. EMR2, differing from CD97 by only three amino acids within the EGF domains, binds CD55 with a Kd at least an order of magnitude weaker than that of CD97 (Lin et al. 2001). Finally, a third ligand of human CD97 was identified by demonstrating that integrin α5β1 (very late antigen [VLA]-5) and possibly also integrin αvβ3 binds the Arg-Gly-Asp (RGD) motif in the stalk region (Wang et al. 2005; compare Fig. 4). The ability of CD97 isoforms, and also other EGF-TM7 receptors, to interact with such a diverse range of ligand types is based on their variable numbers and sequences of N-terminal EGF-like domains (compare Fig. 4). The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with CS (Kwakkenbos et al. 2005).

The majority of such receptor–ligand interactions at the cell surface appear to be weak and transient. Lin and coworkers developed a screening strategy which is specifically suited to identify low-affinity ligands for the EGF-like short consensus repeat domains of EGF-TM7 receptors (Lin et al. 2005). In brief, recombinant expression constructs were engineered encoding the variable EGF-like domains coupled to a mouse Fc fragment and a biotinylation signal sequence. These constructs were then expressed, purified, and biotinylated. Finally, the biotinylated proteins were coupled in a specific orientation to avidin-coated fluorescent beads to screen for ligand-bearing cells or tissues (Lin et al. 2005). Based on these multivalent protein probes, putative cell surface ligands were also identified for other members of the EGF-TM7 clan (Stacey et al. 2001, 2002, 2003).

GPR56, also known as TM7XN1 (7-transmembrane protein with no epidermal growth factor-like NH2-terminal domains 1) functions in tumor cell adhesion and has a role in the development of neural progenitor cells. The amino-terminal domain contains a large number of possible N- and O-linked glycosylation sites similar to mucin-like proteins, but no further adhesion motifs (compare Fig. 1). Tissue transglutaminase 2 (TG2), an integrin-binding adhesion co-receptor for fibronectin, was proposed as a candidate ligand (Xu et al. 2006). Ubiquitously expressed in tissue and tumor stroma, it localizes mainly in the cytoplasm; yet recent reports suggest its presence also at the cell surface, and in the extracellular matrix. TG2 was reported to specifically bind to GPR56 in the mouse (Xu et al. 2006), but not in the human (Nien-Yi Chiang and Hsi-Hsien Lin, personal communication). The significance of this species difference is not known. Although TG2 seemed to interact with the N terminus of mouse GPR56, it is not clear whether it indeed functions as a traditional ligand. As no specific adhesion motifs have been recognized within the N-terminal domain of GPR56, a specific site of interaction remains unknown.

Brain angiogenesis inhibitor 1 (BAI1) is expressed on glial cells within the brain, but also on monocytes and macrophages. Its surface expression is dramatically downregulated in many glioblastomas, consistent with its ability to inhibit angiogenesis and tumor growth in vivo. The large extracellular domain contains one HBD motif and five tandem copies of thrombospondin type 1 repeats (TSRs) (compare Fig. 1). The latter seem to interact with phosphatidylserine (PS), and cells that expressed BAI1 have been shown to selectively engulf a synthetic substrate containing PS. Genetic manipulation of the BAI1 extracellular domain showed that the TSRs were essential for the recognition of PS on apoptotic cells, and direct binding to lipid overlays showed stereospecific binding to PS as well (Park et al. 2007). Also, soluble TSRs derived from BAI1 were shown to inhibit macrophage engulfment of apoptotic thymocytes both ex vivo and in vivo. The adhesive Arg-Gly-Asp (RGD) motif, also present in BAI1, was dispensable under these conditions. Given previous data supporting a role for thrombospondin 1 as a bridging molecule that recognizes apoptotic cells, it may be assumed that PS is a novel ligand for BAI1 that promotes the engulfment activity of cells (see below).

Despite long-standing efforts, no further ligands have as yet been identified for any other members of the adhesion GPCR family. Unavailable 3D structures, heterogeneity of ectodomains, and/or ambiguous relationships within the family make an in silico ligand prediction difficult and error-prone. Although phylogenetically related to the Secretin GPCRs (Nordström et al. 2009), the peptide hormone ligands of the latter receptor family do not provide a clue. Finally, the idea needs to be considered that orphan adhesion GPCRs may have ligand-independent functions. The concept of ligand-independent receptor activation is connected with the observation that some orphan receptors can heterodimerize with structurally unrelated GPCRs which have identified ligands, thereby regulating functions of the latter, while other receptors were shown to be constitutively active without any ligand.

Reassembly of Subunits and Receptor (Hetero-)Oligomerisation

According to the common and well-established paradigm of GPCR activation, ligand binding leads to conformational changes in the heptahelical domain and cytoplasmic tail, thereby activating intracellular signaling pathways. The adhesion GPCRs may be assumed to follow this paradigm. However, the two-subunit configuration with seemingly independent ecto- and endosubunits (“split receptors”; Fig. 3) and receptor dimerisation/ oligomerisation are specific complications which may explain the difficulties in identifying intracellular signaling pathways. Several models are suitable to explain experimental observations concerning subunit interaction and receptor activation (according to Davies et al. 2007; Silva et al. 2009a; Yi-Shu Huang and Hsi-Hsien Lin, personal communication):
  1. (a)

    The mature adhesion GPCR molecule exists as a stable heterodimer. After GPS-cleavage in the ER, the two subunits remain non-covalently bound at the cell surface. Upon ligand binding by the ectosubunit, conformational changes are induced in the non-covalently bound endosubunit, followed by signal transduction into the cell.

     
  2. (b)

    GPS cleavage, possibly in conjunction with additional processing steps, creates two independent molecular entities at the cell surface. Upon ligand binding, the ectoprotein changes its conformation and is enabled to reassociate with the endoprotein, followed by signal transduction into the cell.

     
  3. (c)

    Ectoprotein and endoprotein are independent membrane receptors, each of them binding different ligands. The anchoring mechanism holding the ectoprotein in place is unknown. Upon ligand binding, both membrane proteins are independently capable of signal transduction.

     
  4. (d)

    Ectoprotein and endoprotein are independent membrane proteins. Upon ligand binding, signal transduction into the cell is performed by the endoprotein, while the ectoprotein is shed from the cell surface, possibly serving different biological functions.

     

In the case of CIRL/latrophilin, reassembly of the subunits occurs upon binding of α-latrotoxin to the N-terminal subunit, as suggested in scenario (b); (compare Fig. 3). Subsequently, an intracellular signal is generated via the C-terminus. Thus, the two CIRL/latrophilin fragments do in fact interact; however, the proportion of truly associated, rather than simply colocalized, fragments is difficult to assess (Volynski et al. 2004; Silva et al. 2009a). In C. elegans, constructs expressing an N-terminal lat-1 fragment tethered to the cell surface by a single transmembrane helix instead of the GPS-TM7 regions were not able to complement the lat-1 mutant phenotype (Langenhan et al. 2009). Thus, both subunits seem to be necessary for signaling. The adhesive and signaling properties of Celsr/flamingo, that is, functions of the endo- and ectosubunits, appear to be separated in signaling processes during zebrafish gastrulation (Carreira-Barbosa et al. 2009) as suggested in model (c). This contrasts with results obtained for the lat-1 receptor.

Data presented by Volynski et al. (2004) indicate that agonist-induced dimerisation/ oligomerization of the endoprotein accompanies latrophilin signaling (compare Fig. 3). Similarly, EMR2 is constitutively expressed as a dimer, and the dimerization is mediated exclusively by the heptahelical part (Davies et al. 2007). Interestingly, these adhesion GPCRs can also form hybrid receptor complexes by cross-interaction of heterogeneric receptor subunits. Heterodimerisation between different, but closely related members of the EGF-TM7 family resulted in the modulation of expression and ligand binding properties (Davies et al. 2007). Again, the dimerization seemed to be mediated by the TM7 region and did not involve the posttranslational GPS autoproteolysis. Most recently, “promiscuous” interactions between the endosubunits of completely unrelated receptors were observed, and these cross-complexes seemed to be functionally active, although the in vivo amounts were relatively small (∼10%; Silva et al. 2009a).

Coupling to G Proteins

Activated heptahelical receptors interact with and activate heterotrimeric guanine nucleotide–binding proteins (G proteins) at the inner side of the cell membrane. This association is so well established that the term “G protein-coupled receptor” (GPCR) is used as a synonym. Still, only limited data are available which unequivocally demonstrate G protein-coupling for the adhesion GPCRs. CIRL/Latrophilin/ lectomedin receptor 1 (LEC1/LPHN2) was the first receptor of this group shown to bind to G proteins (Lelianova et al. 1997), specifically Gαq/11 and Gαo (Rahman et al. 1999). The interaction was found to be strong and functional; it was disrupted by conditions that allow G protein activation and dissociation from the receptor (Rahman et al. 1999). The latrophilin–G protein complex was stable in the presence of GDP but dissociated when incubated with GTP, suggesting a functional interaction. Like other receptors, CIRL/Latrophilin was able to activate more than one G protein subtype. As revealed by colocalization studies, it interacted with Gαq/11 and Gαo, but not with Gαs, Gαi, or Gαz, indicating that coupling is specific and not promiscuous. The {alpha}-subunits of Gq and G11 are almost ubiquitously expressed and couple to {beta}-isoforms of phospholipase C (PLC). Activation leads to the production of inositol-1,4,5-trisphosphate causing subsequent release of Ca2+ from intracellular Ca2+ stores (for review, see Silva et al. 2009b). Despite these reports, G protein-coupled signaling of latrophilin/LEC1 remains controversial. A review by Foord et al. (2002) reported that α-latrotoxin did not activate latrophilin/LEC1 in the classical sense as it is still effective when the transmembrane domains of the receptor had been removed. This may be explained by the fact that the toxin itself has the ability to form membrane pores, and that these pores are permeable to cations, especially Ca2+, bypassing any signaling that can be triggered by the receptor. Thus, many questions concerning CIRL signaling remain in the absence of its endogenous ligand(s).

G protein-coupling was also reported for GPR56 (Little et al. 2004; Iguchi et al. 2008), but again no final conclusion can be drawn regarding a general principle of signaling. Mass spectrometry screening in retinoic acid–differentiated NT2 teratocarcinoma cells suggested that GPR56 specifically associates with Gαq/11 as part of a larger complex with tetraspanins CD9 (Tspan29) and CD81 (Tspan28) (Little et al. 2004). These authors at the same time reported a lack of Gαq/11 association for CD97. In an overexpression system, Iguchi et al. (2008) observed that GPR56 signaled via a Gα12/13 and Rho pathway. GPR56-mediated intracellular Ca2+ mobilization, on the other hand, was not observed. G proteins G12 and G13 are often activated by receptors which also couple to Gq/G11. A well-established downstream effector of G alpha 12/13-mediated signaling is the monomeric GTPase RhoA, which is a regulator of actin stress fibers and assembly of focal adhesions, gene transcription, and control of cell growth. Indeed, ectopic expression of GPR56 in NIH3T3 cells induced F-actin accumulation in a Gα12/13- and Rho-dependent manner (Iguchi et al. 2008). The transcriptional activation and actin reorganization were found to be inhibited by an RGS domain of the p115 Rho-specific guanine nucleotide exchange factor (p115 RhoGEF RGS) and a dominant negative form of Rho (Iguchi et al. 2008).

Monk et al. (2009) proposed that DREG/Gpr126 drives the differentiation of promyelinating Schwann cells by elevating intracellular cAMP levels. Since many GPCRs induce the production of cAMP by adenylate cyclase activation, the logical supposition would be that Gpr126 may likewise act as an upstream effector of adenylate cyclase. Still, it remains to be demonstrated that Gpr126 actually associates with a G protein complex, or that the regulation of cAMP by Gpr126 is direct. Similarly, a recent study showed that targeted mutation of the very large G protein-coupled receptor 1 (VLGR1), also known as MASS1 or GPR98, resulted in an increase in the expression and in the redistribution of adenylate cyclase 6 in stereocilia of the cochlea (Michalski et al. 2007). The restricted adenylate cyclase immunostaining just nearby the ankle-link molecular complex (ALC) in wild-type mice and its spreading out along the stereocilia in knockout mice argue in favor of a functional coupling between Vlgr1 and adenylate cyclase 6. Vlgr1 is thus expected to activate the Gαs subunit, which in turn activates cAMP-dependent signaling pathways via adenylate cyclase and protein kinase A (PKA) activation. A-kinase anchoring proteins (AKAPs) are believed to localize PKA to the GPCR-associated molecular complex. Again, however, additional studies showing specific interactions are clearly warranted.

For some other adhesion GPCRs, G protein-coupling is described in the patent literature. Summarizing such patent data, Foord et al. (2002) reported that CD97, EMR1, and HE6/GPR64 will activate the G-proteins Gαs/Gq when overexpressed in Xenopus melanophores (C. Jayawickreme, personal communication). These data seem to be at odds with the reported lack of Gαq/11 association for CD97 by Little et al. (2004). However, the signaling properties of GPCRs may depend on the cellular context. According to Foord et al. (2002), CD55/DAF interaction with CD97 (see above) did not appear to induce any G-protein signaling. Thus, whether signaling after EGF-TM7 ligand interaction occurs via classical heterotrimeric G proteins or uses alternative G-protein-independent signaling pathways remains to be seen. Increasing evidence indicates that many heptahelical receptors, including the adhesion GPCRs, signal through G protein-independent pathways, involving JAK/STATs, Src-family tyrosine kinases, GRKs/β-arrestins, and PDZ domain-containing proteins (see below).

Interactions with Other Proteins

Apart from G protein-coupling, GPCRs can associate with a variety of interacting proteins, such as the β-arrestins, PDZ(PSD95/Dlg/ZO-1), SH2, and polyproline-binding proteins. These GPCR interacting proteins (GIPs) may help to create signaling specificity by engaging additional signaling pathways or localizing signaling events to specific subcellular sites. The predominant target of GIPs is the intracellular C-terminus of GPCRs. PDZ domain proteins, first discovered in the postsynaptic density 95 (PSD95), disk-large (Dlg) and zona occludens-1 (ZO-1) proteins, constitute the largest protein family among the GIPs. They primarily bind a C-terminal S/TXV(L/I) consensus motif, also called a PDZ “ligand.” The ability of PDZ domain proteins to bind short and extreme C-terminal sequences offers a way to interact with target proteins without disrupting their overall structure and function. Their predominant function as GIPs is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs, but they may also regulate the function of their ligands.

A number of adhesion GPCRs express a PDZ recognition motif at their extreme C-terminus, including CD97, BAI1–3, CIRL1/2, VLGR1, as well as GPR123, GPR124, GPR125, and GPR133 (for review, see Bjarnadottir et al. 2007). The consensus motif often is conserved between species, suggesting a functional role. Physical interaction of C-termini with PDZ domain proteins has been described for a several adhesion GPCRs by means of a yeast two-hybrid screen. Using the C-terminus of the receptor molecule as bait, PDZ domain proteins of the Shank family were identified as binding partners of the G protein-coupled α-latrotoxin receptor CL1 (Tobaben et al. 2000, 2002). Shank proteins are multidomain scaffold proteins of the postsynaptic density, connecting neurotransmitter receptors and other membrane proteins with signaling proteins and the actin cytoskeleton (see below). Correspondingly, Kreienkamp et al. (2002) identified the intracellular C-termini of CIRL1 and CIRL2 as interaction partners of the PDZ domain of the proline-rich synapse-associated protein (ProSAP)/somatostatin receptor-interacting protein (SSTRIP) family of postsynaptic proteins (SSTRIP, ProSAP1, and ProSAP2, also known as shank1–shank3, respectively). Shank proteins colocalized with latrotoxin binding GPCR latrophilin 1 (LPHN1; also known as CL1 and CIRL1) at synapses in native brain tissue and may induce clustering of latrophilin 1 in membrane-associated signaling complexes.

The clade of BAI receptors, on the other hand, did not bind to ProSAP/shank (Kreienkamp et al. 2002). Rather, a novel protein was cloned by a similar approach, BAP1 (BAI1-associated protein), which interacts with the cytoplasmic region of BAI1 (Shiratsuchi et al. 1998). The interaction was mediated by the PDZ recognition motif in the carboxy-terminal region of the BAI1 receptor and the PDZ domains of BAP1. BAP1 is a member of the MAGUK (membrane-associated guanylate kinase homologue) family; it possesses a guanylate kinase domain, WW domains, and multiple PDZ domains. The purpose of the interaction is believed to be several fold: (1) the targeting of BAI receptors to their sites of action (e.g., synaptic membranes), (2) anchoring to the actin-based cytoskeleton (see below), and (3) physical association of BAI receptors with elements of the signal transduction machinery (Shiratsuchi et al. 1998).

VLGR1 is involved in the Usher syndrome, an autosomal recessive disorder characterized by combined hearing loss and retinal degeneration. The extreme C-terminus also corresponds to the consensus motif that is recognized as a ligand for the class I subfamily of PDZ domains. Yeast two hybrid and in vitro protein association experiments have shown direct physical interactions between the C-terminus of VLGR1 and the PDZ domain-containing submembrane protein whirlin (Michalski et al. 2007). The large transmembrane protein usherin, the putative transmembrane protein vezatin, and whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells; they are absent in Vlgr1 knockout mice that lack the ankle links (Michalski et al. 2007). The data support the existence of an ankle-link molecular complex (ALC) in the cochlea that includes VLGR1, usherin, vezatin, and whirlin. As all of these proteins bind to myosin VIIa, Michalski et al. (2007) suggested that this actin-based motor protein conveys both transmembrane and submembrane ALC proteins to the stereocilia of the inner ear.

Interactions of adhesion GPCRs and PDZ domain-containing scaffolding molecules with filamentous (F)-actin, as indicated in the examples above, suggest a role in the maintenance and remodeling of the actin cytoskeleton. CIRL-binding ProSAP/shank interacts with fodrin and cortactin–F-actin-binding proteins enriched at cell-matrix contact sites (Kreienkamp et al. 2002). VLGR1 is indirectly connected via its C-terminus to the actin cytoskeleton of stereocilia through the motor domain of myosin VIIa dimers. Similarly, HE6/GPR64 and CD97 colocalize with F-actin in the stereocilia of male excurrent duct epithelia (Kirchhoff et al. 2008; Veninga et al. 2008). In intestinal epithelial cells, CD97 is located in E-cadherin-based adherens junctions and seems to regulate epithelial strength (Becker et al. 2010). Partial co-staining of CD97 and cortical F-actin indicated that the receptor might be involved in anchoring adherens junction components to the cytoskeletal network. The related EMR2 receptor colocalizes with Rac1 (Yona et al. 2008b), a small Rho-GTPase which regulates actin polymerization. When leukocytes were treated for a short period with small amounts of N-formyl-methionine-leucine-phenylalanine, a peptide chain produced by some bacteria, EMR2 and Rac1 were rapidly translocated to the leading edge and other lamellipodia and colocalized with F-actin (Yona et al. 2008b).

ELMO/Dock180/Rac proteins comprise a conserved signaling module which promotes the internalization of apoptotic cells; ELMO and Dock180 function together as a Guanine nucleotide Exchange Factor (GEF) for Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. Using yeast two-hybrid screening to identify upstream ELMO-interacting proteins, Park et al. (2007) identified the BAI1 receptor. Mutational analysis showed that ELMO1 bound to a short alpha-helical stretch within the BAI1 cytoplasmic tail, which was necessary and sufficient for ELMO binding. Furthermore, the formation of a trimeric complex of BAI1–ELMO–Dock180 was associated with enhanced Rac–GTP levels and the greatest increase in apoptotic cell uptake (Park et al. 2007). In addition, the cytoplasmic tail of BAI1 interacts with the Src homology 3 (SH3) domain of a BAI-associated protein 2 (BAIAP2; Oda et al. 1999), also known as the insulin receptor tyrosine kinase substrate of 53 kDa (IRSp53).

GPR56 associates in a complex with Gαq and tetraspanins (Little et al. 2004). Tetraspanin-associated microdomains also connect to the actin cytoskeleton, regulating cell motility and polarity. The C-termini of CD81 and CD9 both possess potential PDZ-domain-binding sites and thus could indirectly link GPR56 to the actin cytoskeleton through the PDZ domains of as yet unknown intracellular proteins. Together with the observation of Rho-dependent actin reorganization during GPR56 signaling (Iguchi et al. 2008), a general role in the dynamic reorganization of the actin cytoskeleton seems likely.

Summary

Although molecular biological and bioinformatics techniques made the identification of all human adhesion GPCRs amenable, the vast majority are still poorly studied orphans with largely unknown structures and functions. Thus, the most important limitation in our current understanding is the persistent paucity of data concerning adhesion GPCR signaling. Being aware of this limitation, the chapter made an effort to list known ligands and to summarize aspects of signal transduction of individual family members in comparison with canonical GPCRs. However, any general characteristics of adhesion GCPR ligands, as well as a generally valid mechanism of receptor docking, ligand-induced activation, signal transduction, and receptor desensitization has remained undiscerned. There are several issues which may account for this shortage. (1) Ligands may be exogenous or may be only expressed in a specific tissue at a particular time under distinct conditions, and a better knowledge of the biology of such receptor/ligand pairs is required before an effort of deorphanization can be undertaken. (2) Orphan adhesion GPCRs may function in the absence of receptor occupation, or some orphans do not activate a signaling cascade alone, but only in conjunction with others. Thus, they may not induce their own second messenger pathway but rather modulate that of others. (3) Some adhesion GPCRs may not stimulate but rather inhibit cell signaling. (4) Although some adhesion GPCRs induce second messenger responses via G proteins, there are indications that others may link to different, perhaps unknown signaling pathways. If this were the case, deorphanization of the remaining will have to wait until these pathways have been defined more clearly. Future challenges in common with other orphan receptors thus are to find endogenous ligands, and to elucidate the general mechanisms underlying the signaling and regulation of receptor desensitization. A unique aspect of the adhesion GCPRs which awaits future elucidation is the significance of their complex posttranslational processing. Many aspects of this processing, including glycosylation, cleavage at the GPS, and the role of “split receptors” subunits remain unclear. The fates of the two subunits and their reassociation at the plasma membrane might provide a variety of different signaling mechanisms which are specific for the adhesion GPCRs.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Andrology, Clinic for Dermatology and VenereologyUniversity Hospital Hamburg-EppendorfHamburgGermany
  2. 2.Wellcome Trust Centre for Human GeneticsUniversity of Oxford Roosevelt DriveOxfordUK