Biochemical and Biophysical Research Communications
Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2
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 Gs-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)
- et al.
Latrophilin signaling links anterior-posterior tissue polarity and oriented cell divisions in the C. elegans embryo
Dev. Cell
(2009) - et al.
Mutations in the VLGR1 gene implicate G-protein signaling in the pathogenesis of usher syndrome type II
Am. J. Hum. Genet.
(2004) - 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) - et al.
Cell adhesion receptor GPR133 couples to Gs protein
J. Biol. Chem.
(2011) - et al.
Signaling property study of adhesion G-protein-coupled receptors
FEBS Lett.
(2012) - et al.
The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity
J. Biol. Chem.
(2011) - et al.
A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133
Cell Rep.
(2014) - et al.
Role of epididymal receptor HE6 in the regulation of sperm microenvironment
Mol. Cell Endocrinol.
(2006) - et al.
New approaches for male fertility control: HE6 as an example of a putative target
Mol. Cell Endocrinol.
(2006) - et al.
Functional consequences of naturally occurring DRY motif variants in the mammalian chemoattractant receptor GPR33
Genomics
(2006)
Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins
Trends Pharmacol. Sci.
Novel designer receptors to probe GPCR signaling and physiology
Trends Pharmacol. Sci.
The adhesion GPCR GPR126 has distinct, domain-dependent functions in Schwann cell development mediated by interaction with laminin-211
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.
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