The activation mechanism and antibody binding mode for orphan GPR20

Abstract

GPR20 is a class-A orphan G protein-coupled receptor (GPCR) and a potential therapeutic target for gastrointestinal stromal tumors (GIST) owing to its differentially high expression. An antibody-drug conjugate (ADC) containing a GPR20-binding antibody (Ab046) was recently developed in clinical trials for GIST treatment. GPR20 constitutively activates Gi proteins in the absence of any known ligand, but it remains obscure how this high basal activity is achieved. Here we report three cryo-EM structures of human GPR20 complexes including Gi-coupled GPR20 in the absence or presence of the Fab fragment of Ab046 and Gi-free GPR20. Remarkably, the structures demonstrate a uniquely folded N-terminal helix capping onto the transmembrane domain and our mutagenesis study suggests a key role of this cap region in stimulating the basal activity of GPR20. We also uncover the molecular interactions between GPR20 and Ab046, which may enable the design of tool antibodies with enhanced affinity or new functionality for GPR20. Furthermore, we report the orthosteric pocket occupied by an unassigned density which might be essential for exploring opportunities for deorphanization.

Unveiling the structural mechanisms of nonpeptide ligand recognition and activation in human chemokine receptor CCR8

“…To investigate whether CCR8 has a high level of constitutive activity through G i pathway, we performed bioluminescence resonance energy transfer (BRET) assays ( 23 ) to measure G protein heterotrimer dissociation in the absence of a ligand ( 24 ). The results revealed that CCR8 displayed high constitutive activity, as evidenced by a notable decrease in its BRET signal compared to negative controls of empty vector (mock control) and adenosine A 2A receptor [A 2A R; known to not couple to G i ( 25 )] as well as positive control GPR20 [known to self-activate G i signaling ( 26 )] (fig. S1A).…”

“…The expression and purification of scFv16 were the same as previously described ( 26 ). The codon-optimized nucleotide sequence of scFv16 was synthesized by GenScript and subcloned into an expression vector pFastBac1 with an 8× His tag at the C terminus.…”

Orphan G protein-coupled receptors: the ongoing search for a home

“…Lin et al attribute the basal activity of GPR20 to an unusually coiled N-terminal helix cap on its transmembrane domain. They also report an orthosteric cavity which could be explored for deorphaization efforts considering that GPR20 is a potential biological target for gastrointestinal tumors (Lin et al, 2023). Alternatively, a seemingly constitutive state could also be due to a strongly bound native ligand (Laschet et al, 2018), as illustrated by GPR40, whose ligand cavity is occupied by native fatty acid ligands (Stoddart et al, 2007).…”

Identification of oleic acid as an endogenous ligand of GPR3

“…For example, GPR52 utilizes its extracellular loop 2 (ECL2) as a built-in ligand to activate itself, 17 and GPR20 uses its N-terminal helix to activate the receptor. 18 In the context of the GPR3 thermogenesis study, the researchers suggested that GPR3 might follow a similar mechanism for self-activation. 16 In the meantime, these previous studies do not preclude the existence of an abundant supply of endogenous ligand within cells to promptly bind and activate GPR3 as soon as the receptor is translated from mRNA, in a term that we refer to as “born to be activated”.…”