Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling

Abstract

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα-mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

A Comprehensive Mutagenesis Screen of the Adhesion GPCR Latrophilin-1/ADGRL1

“…Mutant GPCRs are often overexpressed and constitutively active in various cancers ( Dorsam and Gutkind, 2007 , Kan et al., 2010 , O'Hayre et al., 2013 ). Disease-associated mutations were previously reported to change other adhesion GPCR signaling ( Kishore and Hall, 2017 , Purcell et al., 2017 ). We tested cancer-associated mutations of Lphns reported in different types of carcinomas ( Kan et al., 2010 , O'Hayre et al., 2013 ) and found that the cancer-associated mutations affected basal activity and/or peptide response ( Figure 4 E).…”

GPR56 promotes proliferation of colorectal cancer cells and enhances metastasis via epithelial‑mesenchymal transition through PI3K/AKT signaling activation

“…GPCRs have recently become a hot topic in tumor research ( 19 – 21 ). They are reported to be vital regulators of various cellular functions such as cell adhesion, migration, polarity, and guidance.…”

Structural basis for adhesion G protein-coupled receptor Gpr126 function

“…Although their protein architectures remain largely unknown, functional studies have shown that aGPCR ECRs can regulate receptor function and that antibody-like synthetic proteins that target the ECRs can modulate downstream signaling 9,[22][23][24][25] . A current model for aGPCR regulation suggests that transient interactions between the ECR and 7TM directly regulate receptor signaling 9,[22][23][24][25] . There are also numerous reports that aGPCRs use their ECRs to mediate functions in a 7TM-independent manner [26][27][28][29][30] .…”