Defining the role of Polycystin 1 as a G protein coupled receptor in kidney cystogenesis

About This Grant

Affecting over 12 million people worldwide, Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common inherited kidney diseases and is caused primarily by mutations in two genes—PKD1 and PKD2. In the 30 years since the identification of these genes, extensive effort has been made to understand how their protein products function under normal conditions and further, how naturally occurring mutations alter this function in ADPKD. While significant progress has been made to characterize PKD2 as a Ca2+-permeable nonselective ion channel, the function of PKD1 has proven more elusive. PKD1 has been proposed to act as an atypical G protein-coupled receptor (GPCR), but studies to define this function are limited by the absence of a known activating stimulus and lack of a typical 7-transmembrane (7-TM) domain signature present in all known GPCRs. We have discovered secreted WNT proteins as the activating ligands of the PKD1/PKD2 complex, defining this complex as a distinct class of WNT receptors. We asked the question of whether PKD1 functions as a WNT-activated GPCR. Using super-resolution live cell imaging and bioluminescence resonance energy transfer-based G protein biosensors, we find that PKD1 is directly coupled to specific subsets of heterotrimeric G proteins, such as Gαi1-3 and Gαq, but not Gαs or Gα12/13 in a WNT ligand-dependent manner. Specifically, we show that WNT ligands induce: 1) PKD1-mediated dissociation of Gα from Gβγ subunits, 2) time-dependent recruitment of Gα subunits to PKD1, and 3) Gα-GDP to -GTP transition via PKD1. The WNT-induced coupling of PKD1 to Gαi1-3 inhibits basal and forskolin-induced cAMP accumulation. PKD2 has an essential role in enabling PKD1 to function as a GPCR by chaperoning it to the plasma membrane. Naturally occurring mutations in PKD1 or PKD2 that compromise cell surface expression, assembly of the PKD1/PKD2 complex, or disrupt G protein recruitment diminish WNT-induced PKD1-mediated GPCR signaling. These data, which form the premise of this application, define the molecular function of the Polycystin complex and provide a direct mechanistic link of the complex to cAMP metabolism. We propose complementary in vitro and in vivo genetic approaches to take these findings to the next level to determine whether the direct coupling of PKD1 to Gαi and cAMP signaling is critical for the initiation of cystogenesis. Successful completion of the proposed studies will remove previously impossible technical and conceptual roadblocks to determine how naturally occurring mutations alter the function of these complex molecules and lead towards the development of new and more effective treatments for ADPKD.