Constitutive signaling by the C-terminal fragment of polycystin-1 is mediated by a tethered peptide agonist

Abstract

Mutation of the PKD1 gene, encoding polycystin-1 (PC1), is the primary cause of autosomal dominant polycystic kidney disease. PC1 is an 11-transmembrane domain protein that binds and modulates the activity of multiple heterotrimeric G protein families and is thought to function as a non-canonical G protein-coupled receptor (GPCR). PC1 shares a conserved GPCR autoproteolysis inducing [GAIN] domain with the adhesion family of GPCRs, that promotes an auto-catalytic, cis-cleavage at the GPCR proteolysis site (GPS) located proximal to the first transmembrane domain. GPS cleavage divides these receptors into two associated ‘subunits’, the extracellular N-terminal (NTF) and transmembrane C-terminal (CTF) fragments. For the adhesion GPCRs, removal of the NTF leads to activation of G protein signaling as a result of the exposure and subsequent intramolecular binding of the extracellular N-terminal stalk of the CTF, i.e., the tethered cryptic ligand or tethered agonist model. Here, we test the hypothesis that PC1-mediated signaling is regulated by an adhesion GPCR-like, tethered agonist mechanism. Using cell-based reporter assays and mutagenesis of PC1 expression constructs, we show that the CTF form of PC1 requires the stalk for signaling activation and synthetic peptides derived from the PC1 stalk sequence can re-activate signaling by a ‘stalk-less’ CTF. In addition, we demonstrate that ADPKD-associated missense mutations within the PC1 stalk affect signaling and can inhibit GPS cleavage. These results provide a foundation for beginning to understand the molecular mechanism of G protein regulation by PC1 and suggest that a tethered agonist-mediated mechanism can contribute to PKD pathogenesis.SIGNIFICANCE STATEMENTMutations of the PKD1 gene, encoding polycystin-1, are the predominant cause of autosomal dominant polycystic kidney disease (ADPKD), a systemic disease that is the 4th leading cause of kidney failure. Polycystin-1 functions as an atypical GPCR capable of binding or activating heterotrimeric G proteins, which is essential for preventing renal cystogenesis. However, little is known regarding its regulation. Polycystin-1 shares structural features with the Adhesion family of GPCRs. In this work, we combined mutagenesis and cellular signaling assays which demonstrated that constitutive activation of signaling by polycystin-1 involves an Adhesion GPCR-like molecular mechanism. This study provides new knowledge regarding the structure-function relationships of polycystin-1 which will stimulate additional areas of investigation and reveal novel avenues of therapeutic intervention for ADPKD.

Mechanism of Tethered Agonist-Mediated Signaling by Polycystin-1

“…Substitution of N3074 with a similarly polar Gln residue dramatically reduced CTF signaling ability, replicating the results obtained with N3074A. As mentioned above, an ADPKD-associated mutation, N3074K, also inhibits CTF-mediated activation of the NFAT reporter (45), which may indicate that the size of the residue at position 3074 in addition to its polarity is strictly required and further underscores the importance of this residue in 'setting up' the ability of the stalk to efficiently interact with the TOP domain.…”

“…Substitution of any residue within a protein, let alone a protein with an allosteric functional mechanism, has the potential to alter protein structure, and hence function, which emphasizes the need for additional molecular dynamics simulations to better understand the structure-function relationships of important proteins. Shared features between PC1 and the ADGRs, including auto-proteolytic cleavage at the GPS motif and homology of the GAIN domain, led to the proposal and investigation of whether a TA is involved in PC1 CTF-mediated activation of an NFAT luciferase reporter (45). The current study suggests that the PC1 CTF utilizes a novel allosteric activation mechanism for its TA-mediated signaling activation.…”

“…Shared features between PC1 and the ADGRs, including auto-proteolytic cleavage at the GPS motif and homology of the GAIN domain, led to the proposal and investigation of whether a TA is involved in PC1 CTF-mediated activation of an NFAT luciferase reporter (45). The current study suggests that the PC1 CTF utilizes a novel allosteric activation mechanism for its TA-mediated signaling activation.…”

“…In the tethered agonist (TA) model of ADGRs, dissociation of the NTF results in exposure of a short, extracellular 'stalk' at the N-terminus of the CTF subunit which binds the extracellular loops and 7-TM bundle causing conformational changes that activate G protein signaling (41)(42)(43)(44). We recently demonstrated that PC1 utilizes an ADGR-like TA for constitutive activation of an NFAT promoter-luciferase reporter (45). This work showed that the CTF subunit alone has greater signaling ability than full-length PC1, is dependent on the presence of the stalk and is affected by ADPKDassociated missense mutations within the stalk region.…”

Mechanism of tethered agonist-mediated signaling by polycystin-1

“…In the tethered agonist (TA) model of ADGRs, dissociation of the NTF results in exposure of a short, extracellular “stalk” at the N terminus of the CTF subunit which binds the extracellular loops and 7-TM bundle causing conformational changes that activate G protein signaling ( 41 – 44 ). We recently demonstrated that PC1 utilizes an ADGR-like TA for constitutive activation of an NFAT promoter-luciferase reporter ( 45 ). This work showed that the CTF subunit alone has greater signaling ability than full-length PC1, is dependent on the presence of the stalk, and is affected by ADPKD-associated missense mutations within the stalk region.…”