A Tethered Agonist within the Ectodomain Activates the Adhesion G Protein-Coupled Receptors GPR126 and GPR133

Abstract

Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied class of the GPCR superfamily. aGPCRs are involved in many developmental processes, immune and synaptic functions, but the mode of their signal transduction is unclear. Here, we show that a short peptide sequence (termed the Stachel sequence) within the ectodomain of two aGPCRs, GPR126 and GPR133, functions as a tethered agonist. Upon structural changes within the receptor ectodomain, this intramolecular agonist is exposed to the 7-transmembrane helix domain, which triggers G-protein activation. Our studies show high specificity of a given Stachel sequence for its receptor. Finally, the function of Gpr126 is abrogated in zebrafish with a mutated Stachel sequence, and signaling is restored in hypomorphic gpr126 zebrafish mutants upon exogenous Stachel peptide application. These findings illuminate a previously unknown mode of aGPCR activation, and can initiate the development of specific ligands for this currently untargeted GPCR family.

Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

“…Furthermore, because the Stachel is a central part of the GAIN domain, the release of the Stachel from the GAIN domain most likely leads to a collapse of the original conformation, prohibiting reassociation of the Stachel with the NTF. Although the N-terminal residues of the Stachel are the most deeply buried within the GAIN domain (20,36), they are critical for mediating receptor activation (30,32). Thus, transient exposure of the Stachel to activate the 7TM without causing irreversible NTF-CTF dissociation should be an extremely rare event, if not practically impossible.…”

“…4D), showing that their effects on G-protein signaling measured in this assay are independent of autoproteolysis. Finally, to test if the monobodies functioned in an autoproteolysis-independent but Stachel-dependent manner, we mutated a highly conserved Stachel residue, F385, previously shown to be critical for Stachel-mediated activation of several aGPCRs, including GPR56 (30,32). The F385A mutation did not abolish monobody-mediated modulation of GPR56 signaling (Fig.…”

“…In the Stachel-mediated model, the NTF serves as a protective cap for the Stachel and has no direct role in modulating 7TM function. On ligand binding to an N-terminal adhesion domain, the NTF dissociates from the CTF, termed "shedding," exposing the Stachel to function as a "tethered agonist" (30)(31)(32)(33). Key to this model is GAIN domain autoproteolysis, a necessary reaction to precede shedding and Stachel exposure.…”

G Protein-Coupled Receptors in Myelinating Glia

“…Many adhesion GPCRs undergo autoproteolysis at the GPS motif, resulting in a protein that is separated into an N-terminal fragment (NTF) and a C-terminal fragment (CTF) [10,14]. Gpr126/Adgrg6 was discovered to be essential for Schwann cell myelination by a forward genetic screen in zebrafish [15,16], and subsequent work has firmly demonstrated that the CTF of Gpr126/Adgrg6 couples to Gs proteins, elevates 3', 5'-cyclic adenosine monophosphate (cAMP) levels, activates protein kinase A (PKA), and initiates transcription of key regulators (e.g., Oct6/ Pou3f1) of terminal Schwann cell differentiation in zebrafish as well as mammals [16][17][18][19][20][21][22]. In mammals, Gpr126 mutants also present with limb contracture defects [17]; importantly, human mutations in GPR126/ADGRG6 as well as Adenylate cyclase gene 6 (ADCY6, which encodes a cAMP-synthesizing protein) cause limb and myelination defects, highlighting the conserved and critical function of this pathway in the human PNS [23,24].…”

“…Recent work has demonstrated that receptor autocleavage generates a cryptic tethered agonist ligand termed the Stachel sequence [20]. Stachel-mediated activation of the Gpr126/Adgrg6 CTF potently elevates cAMP, and mutational analysis in zebrafish suggested that signaling via the Stachel sequence is both necessary and sufficient for myelination in vivo [20]. However, the Stachel sequence is predicted to be deeply buried within the GAIN domain of Gpr126/Adgrg6, and it is unclear how this agonist might be exposed to the CTF [14].…”