Activation of Adhesion G Protein-coupled Receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES

Abstract

Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel-derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel-derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective.

Keywords: G protein; G protein-coupled receptor (GPCR); NFAT transcription factor; adhesion; cyclic AMP (cAMP); inositol phosphate; peptides; signal transduction.

FIGURE 1.

Signal transduction and activation of GPR110. A, schematic presentation of a prototypical aGPCR structure. Adhesion GPCR share a common domain called the GAIN domain. At the very C-terminal part of this GAIN domain lies the GPS at which many full-length aGPCRs are autoproteolytically cleaved into an N-terminal fragment (NTF) and a C-terminal fragment (CTF). The recently discovered tethered agonist sequence (Stachel, red cylinder and line) is located between the cleavage site of the GPS and the first transmembrane helix. The sequence of each estimated Stachel sequence is given with the core activating region marked in red letters. B, GPR110 is cleaved at the GPS. A construct containing an N-terminal HA tag and a C-terminal mFc tag was used for cleavage analysis. Upon cleavage at the GPS two fragments, NTF (60 kDa) and CTF (38 kDa), occur (top panel). GPR110 is autoproteolytically processed as the NTF can be detected using an anti-HA antibody (bottom panel, black arrowhead). Note that cleavage appears to be incomplete as a second band can be detected (white arrowhead), which is only slightly smaller than a potential full-length construct (98 kDa). A similar construct of GPR115, which contains a non-cleaving GPS, served as control, yielding one band representing the full-length receptor (90 kDa). C, screening for agonistic peptides derived from GPR110 Stachel sequence. A Stachel-derived peptide library of GPR110 was tested in cAMP and NFAT reporter gene assay as described under ”Methods.“ Data are given as x-fold over empty vector (basal pcDps: for cAMP, 5.9 ± 5.1 nm; for luciferase activity, 322.5 ± 95.7 counts). D, peptide-induced activation of GPR110 in cAMP assay is induced by coupling to the G_s protein. Data are given as x-fold over empty vector, which serves as a negative control (empty vector: cAMP: 2.0 ± 0.1 nm). E, different pGPR110 concentrations were tested on empty vector- and GPR110-transfected cells in cAMP assay. Basal empty vector levels were 100.9 ± 84.2 nm. All assay data are given as mean ± S.D. of three independent experiments, each performed in triplicate. Statistics applied compare basal activity of a given construct to peptide-induced activation (see ”Methods“ for further detail).

FIGURE 2.

Signal transduction and activation of aGPCRs GPR115 and GPR116. A, screening for agonistic peptides derived from the GPR116 Stachel sequence. A Stachel-derived peptide library of GPR116 was tested in cAMP and NFAT reporter gene assay. Data are given as x-fold over empty vector (basal pcDps: for cAMP, 5.9 ± 5.1 nm; for luciferase activity, 322.5 ± 95.7 counts). B, concentration-response analysis of Stachel-derived peptides pGPR110 and pGPR116 on their receptor of origin and empty vector control in IP₁ accumulation assay. Data are given as x-fold over empty vector, which serves as a negative control (IP₁ level, 67.5 ± 15.4 nm). C, screening for agonistic peptides derived from GPR115 Stachel sequence. A Stachel-derived peptide library of GPR115 was tested in cAMP, NFAT reporter gene, and IP₁ accumulation assays. Data are given as x-fold over empty vector, which serves as a negative control (basal pcDps: for cAMP, 5.9 ± 5.1 nm; for luciferase activity, 322.5 ± 95.7 counts; for IP₁ level, 94.4 ± 41.7 nm). All assay data are given as mean ± S.D. of three independent experiments, each performed in triplicate. Statistics applied compare basal activity of a given construct to peptide-induced activation (see ”Methods“ for further detail).

FIGURE 3.

Ex vivo analysis of aGPCR activation by Stachel-derived peptides. A, expression of Gpr110, Gpr115, and Gpr116 determined by quantitative RT-PCR in tissues of wt mice. Gpr110 is expressed strongly in renal papilla. Gpr115 is abundant and Gpr116 is detected ubiquitously. Only very low levels of all receptor transcripts are present in seminal vesicles. B, peptide-stimulated cAMP response in renal papilla and lung. Tissues were isolated from wt and Gpr110 knock-out mice and stimulated as described under ”Methods.“ npGPR110 and npGPR116 serves as a control peptides with no agonistic properties, forskolin served as positive control (pc). Basal cAMP levels (untreated) were 2.4 ± 0.6 nm (wt renal papilla), 2.3 ± 0.4 nm (Gpr110^−/− renal papilla), and 17.1 ± 3.6 nm (lung). Statistics compare raw activities (see ”Methods“ for further detail). C, no cAMP response of pGPR110, pGPR115, and pGPR116 at seminal vesicles. Basal cAMP levels (untreated) were 9.0 ± 4.6 nm, forskolin served as positive control (pc). D, pGPR116 activates its receptor GPR116 ex vivo in lung tissue. IP₁ accumulation was measured by employing an ELISA-based IP₁ accumulation assay. The mean IP₁ level of untreated tissue was 729 ± 220 nm. All data are given as mean x-fold over untreated tissue control ± S.D. of at least three independent experiments, each performed in triplicates. Statistics compare raw activities (see ”Methods“ for further detail).

FIGURE 4.

Specific Stachel sequence-derived peptides of subfamily VI aGPCRs are able to activate several receptors within the group in vitro and ex vivo. A and B, pharmacological characterization of activation capabilities of various Stachel sequence-derived peptides on different aGPCRs. A, cAMP assays, and B, IP₁ assays were performed on empty vector (pcDps)-, GPR110-, GPR115-, and GPR116-transfected cells. pGPR110 and pGPR116 can stimulate GPR110 and GPR116. It is noteworthy that GPR116 activation is only detectable in IP₁ assays. In the same assay, GPR115 can be activated through pGPR116 and pGPR115 activates GPR110. Each transfection was stimulated with all peptides. Data are given as x-fold over empty vector without stimulation, which serves as a negative control (pcDps: for cAMP, 53.6 ± 28.9 nm, for IP₁ activity, 67.5 ± 15.4 nm). All assay data are given as mean ± S.D. of three independent experiments, each performed in triplicate. Statistics applied compare basal activity of given construct to peptide-induced activation (see ”Methods“ for further detail). C, schematic depiction of the activating abilities among the peptides of subfamily VI. Open arrow indicates partial activation of GPR110 through pGPR116 and pGPR115. D, pGPR110 activates GPR116 in lung tissue. Wild-type and Gpr110 knock-out mouse tissue were stimulated with respective peptide or negative control peptide npGPR110. IP₁ levels of untreated tissue were 729 ± 220 (wt) and 809 ± 202 nm (Gpr110^−/−). Data are given as mean ± S.D. of at least three independent experiments, each performed in triplicates. Statistics compare raw activities (see ”Methods“ for further detail).

FIGURE 5.

Residues in the Stachel sequence essential for pGPR115 activity. A, functional rescue of pGPR115 by substitution of two amino acids. Subsequent mutagenesis was performed to gradually change pGPR115 into pGPR116. Peptides were analyzed in an NFAT reporter assay toward their potential to activate GPR115. Decisive positions to restore activity are +9 and +10 (S9P, K10D). Data are given as x-fold over empty vector without stimulation, which serves as a negative control (pcDps: 110.9 ± 57.7 counts). B, evaluation of selected peptide mutants in IP₁ assay. Data are given as x-fold over empty vector without stimulation, which serves as a negative control (pcDps: 56.0 ± 27.7 nm). All data represent mean ± S.D. of three independent experiments each performed in triplicate. Statistics applied compare basal activity of given construct to peptide-induced activation (see ”Methods“ for further detail). C, alignment of the cleavage motifs and Stachel sequences of GPR116 and GPR115 in various species. GPR115 displays a distorted cleavage motif throughout evolution. Residues in the Stachel sequence identified as being essential for tethered agonist activity (PDS in GPR116, red box) are subject to alterations in GPR115 in different species.

FIGURE 6.

Activation between different aGPCR subfamilies. A, COS-7 cells were transfected with the given aGPCR and stimulated with 500 μm pGPR64, pGPR110, and pGPR126. Cyclic AMP accumulation is given as x-fold over empty vector, which serves as a negative control (pcDps: 41.1 ± 3.7 nm). B, comparing concentration-response curves between pGPR126 and pGPR64 on GPR126 showed higher potency and efficacy of pGPR126 on its originating receptor. Increasing concentrations of the given peptide were used to stimulate cAMP accumulation. Cyclic AMP accumulation is given as x-fold over empty vector, which serves as a negative control (pcDps: 0.27 ± 0.13 nm). All data represent mean ± S.D. of three independent experiments each performed in triplicate. Statistics applied compare basal activity of given construct to peptide-induced activation (see ”Methods“ for further detail). C, evolutionary conservation of the Stachel sequences C-terminal of the GPS cleavage site in all mouse aGPCRs. Sequences were retrieved from NCBI/GenBank^TM and aligned using Jalview (32).

FIGURE 7.

Phylogenetic distances between GPCR subtypes. Pharmacological receptor subtypes are considered when individual receptors share the same endogenous agonist. For example, acetylcholine activates muscarinic acetylcholine receptor subtypes 1–5 (M1R, M2R, and M5R shown in this tree). In peptide GPCR field, subtypes are also considered when several closely related peptides activate the same individual receptors. For example, different melanocortins and ACTH activate the five melanocortin receptor subtypes 1–5 (MC1R, MC2R, and MC4R shown in this tree). Furthermore, the vasopressin receptor subtypes V1aR and V2R are activated by vasopressin, but also by high concentrations of oxytocin including the oxytocin receptor (OXTR) as subtype into the vasopressin/oxytocin receptor group. To determine the structural divergence of the individual receptors and receptor subtypes a phylogenetic tree was constructed on the basis of the amino acid identity of the respective human, mouse, and chicken receptor orthologues (to assure the same phylogenetic time) using the identity matrix and ClustalW implemented in DNAStar. The branch lengths of the individual receptors and receptor groups in the phylogenetic tree are a measure of their phylogenetic distances. GPCR6A (member of the glutamate receptor family) was used as outgroup. The phylogenetic distances of GPR110 and GPR116 (subfamily VI, red box) as well as of GPR126 and GPR64 (subfamily VIII, blue box) are comparable with those found, e.g. in muscarinic acetylcholine receptors (M1R, M2R, M5R). The phylogenetic distances of the nucleotide receptor subtypes P2RY1 and P2RY12, both sharing ADP as agonist, are as long as the distances between the aGPCR subfamilies VI and VIII. Histamine receptor subtypes do not cluster (compare H1R, H2R, with H4R) but are still considered as receptor subtypes. This indicates that phylogenetic distances are only a weak measure to predict and group receptor subtypes in respect to pharmacological properties. As shown in this study, Stachel sequence-derived peptides can activate members across previously defined subfamilies of aGPCR (2). Their phylogenetic distances of several aGPCR subfamilies are within the range of distances of well established GPCR subtypes. Consequently, current grouping of aGPCR into 8 subfamilies needs to be reconsidered on the basis of functional data.