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A Functional Study of Disease-Causing GNB1 Mutations

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A Functional Study of Disease-Causing GNB1 Mutations A functional study of disease-causing GNB1 mutations Iulia Pirvulescu Supervisor: Dr. Terence E. Hébert Department of Pharmacology and Therapeutics McGill University, Montréal June 2019 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of MASTER OF SCIENCE © Iulia Pirvulescu 2019 Table of Contents Abstract (English) ........................................................................................................................... 1 Résumé (français) ........................................................................................................................... 3 List of Abbreviations ...................................................................................................................... 5 List of Tables and Figures............................................................................................................... 7 Acknowledgements ......................................................................................................................... 8 1 ─ Introduction .............................................................................................................................. 9 1.1 GPCRs................................................................................................................................... 9 1.1.1 Pharmacological relevance ............................................................................................ 9 1.1.2 Structure ......................................................................................................................... 9 1.1.3 Heterotrimeric G protein activation ............................................................................. 10 1.2 Heterotrimeric G proteins ................................................................................................... 11 1.2.1 Canonical functions of G ......................................................................................... 12 1.2.1.1 Intracellular calcium release ..................................................................................... 13 1.2.2 Noncanonical functions of G ................................................................................... 14 1.2.2.1 G1 in the nucleus ..................................................................................................... 15 1.3 Gβ1 mutations ..................................................................................................................... 15 1.3.1 Genetic perspective ...................................................................................................... 17 1.3.2 Clinical phenotypes ...................................................................................................... 19 1.3.3 GNB1 Syndrome .......................................................................................................... 20 1.3.4 Cancer .......................................................................................................................... 21 1.3.5 Pathways affected that have been studied .................................................................... 22 1.4 Thesis objectives and rationale ........................................................................................... 23 2 ─ Materials and Methods ........................................................................................................... 25 Reagents and Antibodies........................................................................................................... 25 Mutagenesis and cloning........................................................................................................... 26 Cell Culture and Transfection ................................................................................................... 28 Immunofluorescence ................................................................................................................. 29 Cell Lysis and Protein sample preparation ............................................................................... 30 Western blotting ........................................................................................................................ 30 Aequorin Assay ......................................................................................................................... 31 Co-Immunoprecipitation ........................................................................................................... 32 Data Analysis ............................................................................................................................ 33 3 ─ Results .................................................................................................................................... 34 3.1 Creating siRNA-resistant constructs expressing Gβ1 ......................................................... 34 3.2 Verifying the expression of the constructs in HEK 293 cells ............................................. 36 3.3 Verifying the resistance to knockdown by RNA interference ............................................ 38 3.4 Knockdown of the endogenous WT-Gβ1 can be rescued by overexpressing its siGβ1- resistant counterpart, in the aequorin assay .............................................................................. 41 3.5 Gβ1 mutations are categorized as causing gains or losses of function in the M3 mAChR pathway ..................................................................................................................................... 43 3.6 Gβ1 interaction with Gq is compromised by some of the mutations ................................ 47 3.7 Gβ1 mutations impair interaction with RNA polymerase II ............................................... 50 3.8 Co-expression of a mutated and a WT allele has limited effects on protein complexes formed by the mutant ................................................................................................................ 53 4 ─ Discussion .............................................................................................................................. 59 4.1 siGβ1-resistance as a model ................................................................................................ 59 4.2 Mapping functional regions on the Gβ1 gene ..................................................................... 60 4.3 Factors affecting the severity of the disease ....................................................................... 66 4.4 Calcium signaling and disease ............................................................................................ 68 4.5 Heterotrimer formation and cancer ..................................................................................... 69 4.6 Transcriptional regulation ................................................................................................... 71 5 ─ Concluding remarks ............................................................................................................... 73 References ..................................................................................................................................... 75 Abstract (English) G protein-coupled receptors (GPCRs) represent the largest class of membrane receptors in eukaryotes. Activation of GPCRs by extracellular signals leads to conformational changes that activate the associated G protein heterotrimer, composed of the G and G subunits. Mutations in the G1 subunit have been implicated with a number of different cancers and neurodevelopmental disorders. We focused on a set of ten germline and somatic de novo G1 point mutations studying their effects in vitro, with a view toward functionally classifying them. Our objectives were to map functional regions, and connect functional outcomes to clinical phenotypes. We hypothesized that disease-causing G1 mutations confer gains or losses of function that impact their interactions with partners and their effects on signalling pathways. We generated flag-tagged, siRNA-resistant versions of wildtype (WT) and mutated G subunits, to study them in a background of reduced endogenous G1 subunit expression. Our first functional investigation was on intracellular calcium release in response to M3- mAChR stimulation, because calcium signaling has been linked to both cancer and neurological disorders. Previous work in the lab has shown that knockdown of the G1 subunit leads to a significant increase in intracellular calcium release in response to carbachol treatment. Expression of the siRNA-resistant WT-G1 rescued the increase in calcium release caused by WT-G1 knockdown. We were able to classify G1 as D76G and I80T resulted in a loss of function, whereas K57E, K78E and K89E exhibited a dominant negative effect, causing changes in baseline calcium signalling. Next, we assessed whether interactions with Gq/11 and RNA polymerase II (RNAPII) are affected by mutations in G1. We used co-immunoprecipitation to identify which mutations 1 physically interact with Gq/11, the principal G subunit implicated in PLC activation. Only G1 mutants I269T, D76G and A11V maintained a strong interaction with Gq/11. This suggests that G1 is involved in the PLC signaling pathway independently of a Gq/11 interaction, which supports previous work which has shown that G1 modulates calcium release by regulating the expression of G4. In fact, G plays an important role in transcription, occupying over 700 promoters and interacting with RNAPII. We used co-immunoprecipitation to verify if the
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