Abstract: FR-PO585
In Vivo Analysis of Polycystin-1: Heterotrimeric G-protein Binding by NanoBiT
Session Information
- Cystic Kidney Diseases: Mechanisms and Models
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Cystic
Authors
- Magenheimer, Brenda S., The University of Kansas Medical Center, Kansas City, Kansas, United States
- Maser, Robin L., The University of Kansas Medical Center, Kansas City, Kansas, United States
Background
Polycystin-1 (PC1) is an atypical GPCR reminiscent of the Adhesion GPCR family. Both families undergo cis-autocatalyzed cleavage to form extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments with an N-terminal CTF stalk acting as a tethered agonist. PC1-G protein regulation is central to cystogenesis, but little is known regarding mechanisms. Previous studies used truncated PC1 C-tail constructs and in vitro methods to show PC1-G protein binding, with little reported for full-length PC1. We are developing in vivo assays to assess PC1-G protein binding/activation. In this initial analysis, we determined the relative G protein binding preference of PC1 CTF and mutants using Nanoluciferase Binary Technology (NanoBiT) technology that relies on reconstitution of Nanoluc subunits, Large BiT (LgB) with Small BiT (SmB), to form an active enzyme with very bright luminescence.
Methods
Gα-LgB expression clones and HEK293-ΔG7 cells (KO of s,s-L, q,11,12,13, z) were provided by Drs. J. Hansen and A. Inoue. The Native Product (NP) version of SmB was fused to the C-terminus of CTF, ‘stalkless’ ΔstCTF and ADPKD mutants, R4136G and L4137P in the G protein activation motif. HEK293T or ΔG7 cells transfected with CTF-NP and Gα-LgB clones were assessed for Nanoluc complementation with a cell-permeable substrate. Controls included FRB-LgB+FKBP-SmB (positive), and empty vector+Gα-LgB or CTF-NP+FRB-LgB (negative). Luminescence was monitored for 40 min after substrate addition. Relative levels of CTF and Gα-LgB proteins were determined by Western blot and area under the curve was normalized to protein expression levels.
Results
NP did not affect expression or signaling of CTF proteins. CTF had a relative binding preference: Gi > Gq > Gs families in both cell lines. In ΔG7 cells, preferential binding by CTF for Gq > G11 and by ΔstCTF for Gq > Gs was lost. Analyses with a subset of Gα-LgB constructs (s, q, i1) in ΔG7 cells showed reduced binding of Gq relative to Gs for both R4136G and L4137P.
Conclusion
PC1 CTF-G protein binding is promiscuous in vivo and is influenced by the stalk and mutations within the G protein activation motif. Preliminary studies hint that PC1-mediated G protein regulation is complex and may be biased by multiple factors. Future experiments will examine binding in ΔG7 cells with pertussis toxin treatment, longer PC1 expression constructs, and additional mutants.
Funding
- NIDDK Support