Regulation of pulmonary surfactant by the adhesion GPCR GPR116/ADGRF5 requires a tethered agonist-mediated activation mechanism

Abstract

The mechanistic details of the tethered agonist mode of activation for the adhesion GPCR ADGRF5/GPR116 has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3

“…The relative contributions and/or necessity of autoproteolytic cleavage and the tethered agonist to aGPCR activity remain an area of active study and substantial contention. Several published studies have attempted to unravel the mechanistic details of aGPCR action in vivo ( 26 , 27 , 28 , 29 ). However, current reports that use mutagenesis to impair tethered agonist activity have largely ignored the effect of mutations on autoproteolytic cleavage.…”

“…Thus, it is possible that an intact aGPCR heterodimer could unmask the TA sufficiently for interaction with the 7 TM, resulting in receptor activation, and this might also occur for an uncleaved construct to an extent sufficient for signaling. A recent report indicates that an uncleaved knock-in construct of ADGRF5 fails to rescue function in vivo ( 27 ). This contrasts with our work using ADGRL2, in which we show that an uncleaved receptor with an intact TA retains intermediate function relative to WT, whereas an uncleaved TA with a dead TA is without function ( 29 ).…”

Biased signalling is structurally encoded as an autoproteolysis event in adhesion G protein‐coupled receptor Latrophilin‐3/ADGRL3

“…In previous reports, the use of functional assays to monitor downstream signalling pathways of aGPCRs provided a divergent portrait of the importance of GPS autoproteolysis for receptor functions 13,29,30 . Moreover, aGPCRs have been shown to couple to various G protein families, in particular ADGRL3, which is able to form stable active complexes with Gi, Gs, Gq and G12/13 protein families 23,24 .…”

“…In previous reports, the use of functional assays to monitor downstream signalling pathways of aGPCRs provided a divergent portrait of the importance of GPS autoproteolysis for receptor functions. 13,29,30 Moreover, aGPCRs have been shown to couple to various G protein families, in particular ADGRL3, which is able to form stable active complexes with Gi, Gs, Gq and G12/13 protein families. 23,24 In this study, we sought to address the impact of autoproteolysis by monitoring the direct functional coupling of ADGRL3-T855G with G proteins, some of the receptor's most upstream effectors (Figure 2A).…”

Biased Signaling is Structurally Encoded As An Autoproteolysis Event in Adhesion G Protein-Coupled Receptor Latrophilin-3/ADGRL3

“…In previous reports, the use of functional assays to monitor downstream signaling pathways of aGPCRs provided a divergent portrait of the importance of GPS autoproteolysis for receptor functions 12,23,24 . Moreover, aGPCRs have been shown to couple to various G protein families, in particular ADGRL3 being able to form stable active complexes with Gi, Gs, Gq and G12/13 proteins families 17,18 .…”