Aberrant expression, function, and mutation of G protein-coupled receptors (GPCRs) and their signaling partners, G proteins, have been well documented in many forms of cancer. These cell surface receptors and their endogenous ligands are implicated in all aspects of cancer including proliferation, angiogenesis, invasion, and metastasis. Adhesion GPCRs (aGPCRs) form the second largest family of GPCRs, most of which are orphan receptors with unknown physiological functions. This is mainly due to our limited insight into their structure, natural ligands, signaling pathways, and tissue expression profiles. Nevertheless, recent studies show that aGPCRs play important roles in cell adhesion to the extracellular matrix and cell−cell communication, processes that are dysregulated in cancer. Emerging evidence suggests that aGPCRs are implicated in migration, proliferation, and survival of tumor cells. We here review the role of aGPCRs in the five most common types of cancer (lung, breast, colorectal, prostate, and gastric) and emphasize the importance of further translational studies in this field.

Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

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Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are required for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that the ECL2 mutation ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

ADGRG2(GPR64) is an orphan adhesion G protein‐coupled receptor (aGPCR), which is expressed in the epididymis and parathyroid glands. We have previously shown that ADGRG2 regulates the secretion of parathyroid hormone by parathyroid adenoma cells via interaction with and altering calcium‐sensing receptor signaling. Generally, dissociation of a cleaved N‐terminal Fragment (NTF) from the C‐terminal fragment is a prerequisite for the activation of aGPCRs. Our previous data confirmed this mechanism in human ADGRG2, as well as the role of b‐arrestins, dynamin, and GPCR kinases in the signaling of NTF‐truncated ADGRG2. However, the structural motifs other than the NTF that regulate signaling and trafficking of ADGRG2 remain unknown. Herein, we mutated residues in the extracellular loop 2 (ECL2) that are conserved among zebrafish, mouse, and human ADGRG2, as well as the whole family of aGPCRs. Using HEK293 cells as a cell model, we elucidated how these mutations alter cAMP production and CRE induction (Biosensor and Luciferase Reporter Assays), cell resistance (label‐free impedance assay), receptor surface expression (ELISA, Immunofluorescence imaging), and receptor degradation (Western blotting). We discovered that mutations in tryptophan and isoleucine of ECL2 significantly decrease surface and total expression of ADGRG2. Treatment with a translation inhibitor, cycloheximide, revealed that these mutants are degraded at a much higher rate compared to the WT receptor. Having adjusted the dose of plasmids to reach similar surface expression, we discovered that ECL2‐mutated receptors do not respond to the activating synthetic peptide, P‐15, in any of the signaling readouts. Using a fluorophore‐conjugated P‐15, we found that ECL2 plays a critical role in the binding of the activating peptide to ADGRG2. These data suggest a major role for ECL2 and its conserved residues in signaling, trafficking, and stability of ADGRG2.