Biology subjects

Magenheimer, B. S.

Publications and source records attributed to Magenheimer, B. S..

3 recordsLinked to original sources

bioRxiv2021

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

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.

biochemistry↗

bioRxiv2021

Mechanism of Tethered Agonist-Mediated Signaling by Polycystin-1

Polycystin-1 (PC1) is an important unusual G-protein-coupled receptor (GPCR) with 11 transmembrane (TM) domains and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal and membrane-embedded, C-terminal (CTF) fragments, and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074-S3585 and R3848-E4078 predicted from the GaMD simulations were validated by new mutagenesis experiments. Together, these complementary analyses have provided novel insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1. Significance StatementMutations of polycystin-1 (PC1) are the major cause (85% of cases) of autosomal dominant polycystic kidney disease (ADPKD), which is the fourth leading cause of kidney failure. PC1 is thought to function as an atypical GPCR, yet the mechanism by which PC1 regulates G-protein signaling remains poorly understood. A significant portion of ADPKD mutations of PC1 encode a protein with defects in maturation or reduced function that may be amenable to functional rescue. In this work, we have combined complementary biochemical and cellular assay experiments and accelerated molecular simulations, which revealed a novel allosteric transduction pathway in activation of the PC1 CTF. Our findings shall facilitate future rational drug design efforts targeting the PC1 signaling function.

biochemistry↗

bioRxiv2020

The Lonidamine Derivative H2-Gamendazole Reduces Cyst Formation in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating renal neoplastic disorder with limited treatment options. It is characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. We have examined the effectiveness of the indazole carboxylic acid, H2-gamendazole (H2-GMZ), a derivative of lonidamine, to inhibit these processes and cyst formation using in vitro and in vivo models of ADPKD. H2-GMZ was effective in rapidly blocking forskolin-induced, Cl--mediated short-circuit currents in human ADPKD cells at 1 M and it significantly inhibited both cAMP- and EGF-induced proliferation of ADPKD cells with an IC50 of 5-10 M. Western blot analysis of H2-GMZ-treated ADPKD cells showed decreased phosphorylated ERK and hyperphosphorylated Rb levels. H2-GMZ treatment also decreased ErbB2, Akt, and Cdk4, consistent with inhibition of the chaperone Hsp90, and reduced the levels of the CFTR Cl- channel. H2-GMZ-treated ADPKD cultures contained a higher proportion of smaller cells with fewer and smaller lamellipodia and decreased cytoplasmic actin staining, and they were unable to accomplish wound closure even at low H2-GMZ concentrations, consistent with an alteration in the actin cytoskeleton and decreased cell motility. Studies using mouse metanephric organ cultures showed that H2-GMZ inhibited cAMP-stimulated cyst growth and enlargement. In vivo, H2-GMZ (20mg/kg) was effective in slowing postnatal cyst formation and kidney enlargement in the Pkd1flox/flox:Pkhd1-Cre mouse model. Thus, H2-GMZ treatment decreases Cl- secretion, cell proliferation, cell motility, and cyst growth. These properties, along with its reported low toxicity, suggest that H2-GMZ might be an attractive candidate for treatment of ADPKD.

pharmacology and toxicology↗