Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2

doi:10.1016/j.bbrc.2015.07.020

Biochemical and Biophysical Research Communications

Volume 464, Issue 3, 28 August 2015, Pages 743-747

https://doi.org/10.1016/j.bbrc.2015.07.020 Get rights and content

Highlights

•
Signal transduction and activation mechanism of the orphan GPR64 were investigated.
•
GPR64 elevates basal cAMP production in transfected cells.
•
GPR64 is specifically activated through a tethered agonist.
•
Activated GPR64 shows promiscuous G-protein coupling.

Abstract

The epididymis-specific adhesion G protein-coupled receptor (aGPCR) GPR64/ADGRG2 has been shown to be a key-player in the male reproductive system. As its disruption leads to infertility, GPR64 has drawn attention as potential target for male fertility control or improvement. Like the majority of aGPCRs GPR64 is an orphan receptor regarding its endogenous agonist and signal transduction. In this study we examined the G protein-coupling abilities of GPR64 and showed that it is activated through a tethered agonist sequence, which we have previously identified as the Stachel sequence. Synthetic peptides derived from the Stachel region can activate the receptor, opening for the first time the possibility to externally manipulate the receptor activity.

Graphical abstract

Introduction

Among the superfamily of G protein-coupled receptors (GPCRs) the class of adhesion GPCRs (aGPCRs) is the second largest [1], [2], yet the most neglected one. Although increasing information about their relevance is available from gene-deficient animals [3], [4], [5], human diseases [6], [7] and variant-associated phenotypes [8], [9], [10] surprisingly little is known about the molecular function of aGPCRs. With up to 6300 amino acids aGPCRs are among the largest proteins in nature composed of a long extracellular domain (ECD), a seven-transmembrane domain (7TM) and an intracellular C-terminal tail (ICD) [11], [12]. A hallmark of this receptor group is the highly conserved GPCR autoproteolysis inducing (GAIN) domain which contains the GPCR proteolysis site (GPS) where the receptor is processed into an N-terminal fragment (NTF) and a C-terminal fragment (CTF) (Fig. 1A).

The mode of signal transduction is an essential piece in understanding the receptor function but this is still unsolved for most aGPCRs. Until recently it was uncertain whether aGPCRs couple to G proteins at all. Recently, more direct evidence for G_s-protein coupling was provided measuring intracellular cAMP levels induced by basal activity of the aGPCRs GPR133 and GPR126 [13], [14], [15]. Increased receptor activity was described after autoproteolytic cleavage at the GPS and removal of the resulting NTF [16], [17], [18] leading to the assumption that the NTF contains an inverse agonist. Using these active CTF mutants fused with the N terminus of the human P2Y₁₂ receptor to ensure membrane expression (Fig. 1A) we have shown that GPR126 and GPR133 contain a tethered peptide agonist in the very N terminus of the CTF [19]. Peptides derived from this region, called Stachel sequence, were able to activate G protein-mediated signal transduction in vitro and in vivo. Recently, the concept of a tethered peptide agonist was independently confirmed on the aGPCR GPR56 and GPR110 [20].

Expression of the orphan GPR64/ADGRG2 is normally restricted to the epididymis [21] where it is essential for maintaining male fertility [22], [23]. This observation has sparked marked interest in this receptor as a potential target for male contraception [24], [25] or fertility improvement. GPR64 was found to be over-expressed in human cancer as in Ewing's sarcoma, where it increases malignancy of the tumor [26], [27]. Therefore, identifying the mode of signaling and modulators of GPR64 activity is of high interest. Here, we characterize the G protein-mediated signal transduction of GPR64 and describe a Stachel sequence-derived agonistic peptide.

Section snippets

Materials

If not stated otherwise, all standard substances were purchased from Sigma–Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), and C. Roth GmbH (Karlsruhe, Germany). Cell culture material and primers were obtained from Invitrogen (Darmstadt, Germany).

Methods

Generation of wild type and mutant GPR64 constructs - Full-length mouse GPR64 (isoform 4: NM_001079848.1) sequence was amplified from mouse testis cDNA library (primer: forward 5′cacacggagtttcctcccta-‘/reverse 5’-tcctttcgaggttgctgaat-3′),

Results

Wildtype and chimeric GPR64 show increased basal activity in G protein-dependent pathways – Clarifying the signal transduction of an unknown GPCR is a central issue in its deorphanization. Despite many efforts over the past years the signaling mode of many aGPCRs including GPR64 remained enigmatic. The enormous size of aGPCRs and the absence of agonists have long hampered efforts to characterize the signaling modes of this receptor class. Based on the commonly accepted concept in which GPCRs

Discussion

GPR64 is a receptor with strong clinical implications as it is tightly linked to male fertility and cancer progression. Considering that GPCRs are in general ideal targets for pharmaceutical treatment, deciphering the signal transduction and activation mode of GPR64 is a decisive step towards future therapeutic applications. We show that GPR64 signals via downstream effectors of Gs and Gi in addition to its already identified Gq-protein coupling [22]. However, in our experimental setup the full

Acknowledgments

We are grateful to Kay-Uwe Simon for technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft [FOR2149] and the Bundesministerium für Forschung und Bildung (IFB AdipositasDiseases Leipzig [K7-67]).

References (40)

T. Langenhan et al.
Latrophilin signaling links anterior-posterior tissue polarity and oriented cell divisions in the C. elegans embryo
Dev. Cell
(2009)
M.D. Weston et al.
Mutations in the VLGR1 gene implicate G-protein signaling in the pathogenesis of usher syndrome type II
Am. J. Hum. Genet.
(2004)
R.C. Harvey et al.
Identification of novel cluster groups in pediatric high-risk B-precursor acute lymphoblastic leukemia with gene expression profiling: correlation with genome-wide DNA copy number alterations, clinical characteristics, and outcome
Blood
(2010)
J. Bohnekamp et al.
Cell adhesion receptor GPR133 couples to Gs protein
J. Biol. Chem.
(2011)
J. Gupte et al.
Signaling property study of adhesion G-protein-coupled receptors
FEBS Lett.
(2012)
K.J. Paavola et al.
The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity
J. Biol. Chem.
(2011)
I. Liebscher et al.
A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133
Cell Rep.
(2014)
C. Kirchhoff et al.
Role of epididymal receptor HE6 in the regulation of sperm microenvironment
Mol. Cell Endocrinol.
(2006)
U. Gottwald et al.
New approaches for male fertility control: HE6 as an example of a putative target
Mol. Cell Endocrinol.
(2006)
H. Rompler et al.
Functional consequences of naturally occurring DRY motif variants in the mammalian chemoattractant receptor GPR33
Genomics
(2006)

R.J. Lefkowitz et al.

Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins

Trends Pharmacol. Sci.

(1993)

J. Wess et al.

Novel designer receptors to probe GPCR signaling and physiology

Trends Pharmacol. Sci.

(2013)

S.C. Petersen et al.

The adhesion GPCR GPR126 has distinct, domain-dependent functions in Schwann cell development mediated by interaction with laminin-211

Neuron

(2015)

T.K. Bjarnadottir et al.

The adhesion GPCRs: a unique family of G protein-coupled receptors with important roles in both central and peripheral tissues

Cell Mol. Life Sci.

(2007)

J. Hamann et al.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors

Pharmacol. Rev.

(2015)

H. Waller-Evans et al.

The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse

PLoS One

(2010)

K.R. Monk et al.

A G protein-coupled receptor is essential for Schwann cells to initiate myelination

Sci. (New York, N.Y.)

(2009)

X. Piao et al.

G protein-coupled receptor-dependent development of human frontal cortex

Science

(2004)

F. Marroni et al.

A genome-wide association scan of RR and QT interval duration in 3 European genetically isolated populations: the EUROSPAN project

Circ. Cardiovasc Genet.

(2009)

A. Tonjes et al.

Genetic variation in GPR133 is associated with height: genome wide association study in the self-contained population of Sorbs

Hum. Mol. Genet.

(2009)

Cited by (83)

Intramolecular activity regulation of adhesion GPCRs in light of recent structural and evolutionary information
2023, Pharmacological Research
The class B2 of GPCRs known as adhesion G protein-coupled receptors (aGPCRs) has come under increasing academic and nonacademic research focus over the past decade due to their physiological importance as mechano-sensors in cell-cell and cell-matrix contexts. A major advance in understanding signal transduction of aGPCRs was achieved by the identification of the so-called Stachel sequence, which acts as an intramolecular agonist at the interface between the N terminus (Nt) and the seven-transmembrane helix domain (7TMD). Distinct extracellular signals received by the Nt are integrated at the Stachel into structural changes of the 7TMD towards an active state conformation. Until recently, little information was available on how the activation process of aGPCRs is realized at the molecular level. In the past three years several structures of the 7TMD plus the Stachel in complex with G proteins have been determined, which provide new insights into the architecture and molecular function of this receptor class. Herein, we review this structural information to extract common and distinct aGPCR features with particular focus on the Stachel binding site within the 7TMD. Our analysis extends the current view of aGPCR activation and exposes similarities and differences not only between diverse aGPCR members, but also compared to other GPCR classes.
GPR114/ADGRG5 is activated by its tethered peptide agonist because it is a cleaved adhesion GPCR
2023, Journal of Biological Chemistry
Family B2 or adhesion G protein–coupled receptors (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR autoproteolysis-inducing domain that self-cleaves the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), or seven transmembrane domain (7TM). The NTF and CTF remain bound after cleavage through noncovalent interactions. NTF binding to a ligand(s) presented by nearby cells, or the extracellular matrix anchors the NTF, such that cell movement generates force to induce NTF/CTF dissociation and expose the AGPCR tethered peptide agonist. The released tethered agonist (TA) binds rapidly to the 7TM orthosteric site to activate signaling. The orphan AGPCR, GPR114 was reported to be uncleaved, yet paradoxically capable of activation by its TA. GPR114 has an identical cleavage site and TA to efficiently cleave GPR56. Here, we used immunoblotting and biochemical assays to demonstrate that GPR114 is a cleaved receptor, and the self-cleavage is required for GPR114 TA-activation of Gs and no other classes of G proteins. Mutagenesis studies defined features of the GPR114 and GPR56 GAIN_A subdomains that influenced self-cleavage efficiency. Thrombin treatment of protease-activated receptor 1 leader/AGPCR fusion proteins demonstrated that acute decryption of the GPR114/56 TAs activated signaling. GPR114 was found to be expressed in an eosinophilic-like cancer cell line (EoL-1 cells) and endogenous GPR114 was efficiently self-cleaved. Application of GPR114 TA peptidomimetics to EoL-1 cells stimulated cAMP production. Our findings may aid future delineation of GPR114 function in eosinophil cAMP signaling related to migration, chemotaxis, or degranulation.
Crystal Structure of the Extracellular Domains of GPR110
2023, Journal of Molecular Biology
Adhesion G protein-coupled receptors (aGPCRs) play a pivotal role in human immune responses, cellular communication, organ development, and other processes. GPR110 belongs to the aGPCR subfamily VI and was initially identified as an oncogene involved in lung and prostate cancers. GPR110 contains tandem adhesion domains at the extracellular region that mediate inter-cellular signaling. However, the structural organization and signaling mechanism for these tandem domains remain unclear. Here, we report the crystal structure of a GPR110 fragment composing the SEA, HormR, and GAIN domains at 2.9 Å resolution. The structure together with MD simulations reveal rigid connections between these domains that are stabilized by complementary interfaces. Strikingly, we found N-linked carbohydrates attached to N389 of the GAIN domain form extensive contacts with the preceding HormR domain. These interactions appear to be critical for folding, as removal of the glycosylation site greatly decreases expression of the GPR110 extracellular fragment. We further demonstrate that the ligand synaptamide fits well within the hydrophobic pocket occupied by the Stachel peptide in the rest state. This suggests that the agonist may function by removing the Stachel peptide which in turn redocks to the orthosteric pocket for receptor activation. Taken together, our structural findings and analyses provide novel insights into the activation mechanism for aGPCRs.
Adhesion G protein-coupled receptors—Structure and functions
2023, Progress in Molecular Biology and Translational Science
Adhesion G protein-coupled receptors (aGPCRs) are an ancient class of receptors that represent some of the largest transmembrane-integrated proteins in humans. First recognized as surface markers on immune cells, it took more than a decade to appreciate their 7-transmembrane structure, which is reminiscent of GPCRs. Roughly 30 years went by before the first functional proof of an interaction with a G protein was published. Besides classic features of GPCRs (extracellular N terminus, 7-transmembrane region, intracellular C terminus), aGPCRs display a distinct N-terminal structure, which harbors the highly conserved GPCR autoproteolysis-inducing (GAIN) domain with the GPCR proteolysis site (GPS) in addition to several functional domains. Several human diseases have been associated with variants of aGPCRs and subsequent animal models have been established to investigate these phenotypes. Much progress has been made in recent years to decipher the structure and functions of these receptors. This chapter gives an overview of our current understanding with respect to the molecular structural patterns governing aGPCR activation and the contribution of these giant molecules to the development of pathologies.
Activation of the adhesion G protein-coupled receptor GPR133 by antibodies targeting its N-terminus
2022, Journal of Biological Chemistry
We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein–coupled receptor involved in raising cytosolic cAMP levels, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. Our previous work suggested that dissociation of autoproteolytically generated N-terminal and C-terminal fragments of GPR133 at the plasma membrane correlates with receptor activation and signaling. To promote the goal of developing biologics that modulate GPR133 function, we investigated the effects of antibodies against the N-terminus of GPR133 on receptor signaling. Here, we show that treatment of HEK293T cells overexpressing GPR133 with these antibodies increased cAMP levels in a concentration-dependent manner. Analysis of culture medium following antibody treatment further indicated the presence of complexes of these antibodies with the autoproteolytically cleaved N-terminal fragments of GPR133. In addition, cells expressing a cleavage-deficient mutant of GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage dependent. Finally, we demonstrate the antibody-mediated stimulation of WT GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that the intramolecular cleavage in the N-terminus modulates receptor activation and signaling.
SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic β-cells
2021, Molecular Metabolism
Members of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear.
Here, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 β-cells and in MIN6 β-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and β-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 β-cells and human islets.
SNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 β-cells did not affect insulin secretion. However, it was associated with reduced β-cell apoptosis, while the deletion of GPR56 made MIN6 β-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 β-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect.
These data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in β-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect β-cells against apoptosis, offering a potential therapeutic target to maintain β-cell mass in type 2 diabetes.

View all citing articles on Scopus

View full text

Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Methods

Results

Discussion

Acknowledgments

Dev. Cell

Am. J. Hum. Genet.

Blood

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Cell Rep.

Mol. Cell Endocrinol.

Mol. Cell Endocrinol.

Genomics

Trends Pharmacol. Sci.

Trends Pharmacol. Sci.

Neuron

The adhesion GPCRs: a unique family of G protein-coupled receptors with important roles in both central and peripheral tissues

Cell Mol. Life Sci.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors

Pharmacol. Rev.

The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse

PLoS One

A G protein-coupled receptor is essential for Schwann cells to initiate myelination

Sci. (New York, N.Y.)

G protein-coupled receptor-dependent development of human frontal cortex

Science

A genome-wide association scan of RR and QT interval duration in 3 European genetically isolated populations: the EUROSPAN project

Circ. Cardiovasc Genet.

Genetic variation in GPR133 is associated with height: genome wide association study in the self-contained population of Sorbs

Hum. Mol. Genet.