Cryo‐EM structure of orphan G protein‐coupled receptor GPR21

Abstract

GPR21 belongs to class A orphan G protein‐coupled receptor (GPCR). The endogenous ligands for human GPR21 remain unidentified. GPR21 expression is associated with developing type 2 diabetes (T2DM), a multifactorial metabolic disease caused by pancreatic β‐cell dysfunction, decreasing insulin production, insulin resistance, and obesity. Animal studies suggested that GPR21 is a potential therapeutic target for T2DM treatment. The underlying mechanisms leading to GPR21 self‐activation remain unknown. In our co‐expression analysis, we noted that GPR21 could also form a stable complex with an unreported Gα protein subtype, Gαs. To gain further insights into the structural mechanisms of GPR21 activation, we employed cryo‐electron microscopy (cryo‐EM) and single‐particle analysis to resolve the high‐resolution structure of GPR21‐Gαs complexes. The clear electron density map of the GPR21‐Gαs provided direct evidence that GPR21 could couple to Gαs protein at physiological conditions. Thus, GPR21 might mediate previously unexplored pathways in normal or pathological conditions, which warrants further investigation. Structure‐guided mutagenesis and biochemical analysis revealed that extracellular loop 2 (ECL2) of GPR21 is essential for the receptor transducing intracellular signal via cAMP. Together, the new structure data reveal a novel signaling cascade of human GPR21 mediated by ECL2 and provide fundamental information for future structure‐based drug development.

An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism

“…Unexpectedly, a disulfide observed between TM6 and TM7, while adjacent to the critical tryptophan rotamer toggle switch, proved not to be critical for GPR61 constitutive activity. The agonist-like motif (ALM) of ECL2 reported for GPR52, GPR21, and GPR12 30 – 33 to drive constitutive activity also does not appear to be present in GPR61, and we have found no structural evidence of binding to the orthosteric site by N-terminal residues with a reported role in activation 9 . The lack of some key sequence features found in many other receptors, such as an ionic lock or sodium binding site, may suggest less stabilization of the inactive state.…”

“…An agonist-like motif (ALM) found in ECL2 has recently been identified in several receptors, including GPR52, GPR21, and GPR12 30 – 33 as a stimulator of constitutive receptor activity. To probe the potential role of ECL2 in GPR61 activation, we examined the basal activity of GPR61 with point mutations in ECL2 designed to weaken its interactions with the orthosteric site (Q198A, W199A) and/or abolish the conserved disulfide (Δ195-201(GGSGGSGG), C195A) (Supplementary Fig.…”

Molecular features of the ligand-free GLP-1R, GCGR and GIPR in complex with Gs proteins

“…4d ). Recent studies have shown that several class A orphan GPCRs are self-activated by ECL2 that directly penetrates to the orthosteric binding pocket, such as GPR21 22 , GPR52 23 , GPR17 24 , GPR161 and GPR61 25 . Although the ECL2 of GCGR partially overlaps with the peptide-binding site, the majority of which is likely disordered owing to the missing cryo-EM density, suggesting its conformational flexibility and non-built-in agonist nature.…”

Specific binding of GPR174 by endogenous lysophosphatidylserine leads to high constitutive Gs signaling

“…Recently published structures of GPR12 11 in subgroup II and GPR21 9 , 10 and GPR52 8 in subgroup III in complex with G s revealed no ligands bound to the receptors, but instead showed that ECL2 of these receptors penetrate into the orthosteric pocket, leading to a self-activation model. Since no structures of receptors in group I have been reported, we further determined the cryo-EM structures of GPR161‒G s and GPR61‒G s complexes using the same strategy as GPR174 at nominal resolutions of 3.1 and 3.2 Å, respectively (Supplementary Figs.…”