Maser Laboratory: Research Projects
Eighty-five percent of the cases of ADPKD are due to mutation of the PKD1 gene, which encodes polycystin-1 (PC1), a large and multi-faceted protein thought to function as an atypical G protein-coupled receptor (GPCR). We, and others, have recently shown that the loss of PC1-G protein regulation is fundamental to the pathogenesis of PKD. However, very little is currently known regarding the mechanism whereby PC1 regulates G protein activity.
Our long-term goal is to understand the structure-function relationships underlying this crucial function and how its dysregulation leads to disease pathogenesis in order to target and functionally restore or augment PC1 function as a treatment for ADPKD.
PC1 shares both structural and functional features with the adhesion class of GPCRs, most notably an extracellular, N-terminal GAIN domain that is responsible for an auto-catalytic, cis-cleavage event that occurs at a specific site (the GPS) within its 3-D structure. Cleavage at the GPS generates two fragments of the receptor, the N-terminal (NTF) and C-terminal (CTF) fragments, which remain non-covalently attached. For a number of adhesion GPCRs, GPS cleavage followed by dissociation of the NTF and CTF portions, due to ligand binding by the NTF or mechanical forces, leads to the liberation of the N-terminal end of the CTF stalk, called the Stachel sequence, that then functions as a tethered, ‘de-crypted’ ligand by binding to the remainder of the CTF and activating G protein signaling. For PC1, GPS cleavage is known to be crucial for its functionality and for the prevention of PKD. We have proposed that a mechanism similar to the adhesion GPCRs is involved in regulation of G protein signaling by PC1 (Fig 1).
The major goals of our current research are to decipher the mechanism of PC1 stalk/Stachel sequence-mediated regulation of G protein signaling and to demonstrate the relevance of the Stachel-like sequence and stalk region of PC1 in the pathogenetic mechanism and potential treatment of PKD.
Specifically, we are working to determine the essential properties of the tethered peptide ligand of PC1 responsible for its regulation of G protein signaling, map the regions of PC1 involved in tethered ligand-dependent signaling, and determine the conditions for the peptide ligand-dependent rescue of renal cystogenesis.
We employ transfection of established cell lines along with standard molecular biology methods and biochemical assays to assess the signaling capability of modified expression constructs of PC1 and of soluble tethered ligand-derived peptides.
We utilize the metanephric organ culture method for treatment of Pkd1 mutant kidneys with soluble tethered ligand-derived peptides as a more physiological model system in order to optimize stalk peptides and determine their effects on cystogenesis.
We have also teamed up with Yinglong Miao's lab at KU-Lawrence, expert in molecular dynamic simulations with biomolecules, to understand the dynamic structure-function relationships for PC1-mediated G protein regulation.
Elucidating the mechanism of PC1-regulated G protein signaling will advance our understanding of the molecular pathogenesis of ADPKD, which is imperative for the development of new therapies for the treatment or prevention of this disease.