Analysis of Naturally Occurring Genetic Variants in the Sphingosine-1-Phosphate Receptor Family
Open Access
- Author:
- Hornick, Jacob
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 27, 2019
- Committee Members:
- James Riley Broach, Dissertation Advisor/Co-Advisor
James Riley Broach, Committee Chair/Co-Chair
Ralph Lauren Keil, Committee Member
Ira Joseph Ropson, Committee Member
Richard Bernard Mailman, Outside Member - Keywords:
- Personalized medicine
GPCR
S1P
RRMS
G protein
Genetic variation
yeast - Abstract:
- The field of personalized medicine has significantly expanded with the advent of gene sequencing and the decreasing cost and increasing availability of whole genome sequencing data. This dissertation focused on a family of sphingosine-1-phosphate receptors, members of the G protein-1-coupled receptor (GPCR) superfamily. GPCRs, have been of interest because they are the largest superfamily in the human genome, occur across species, and play a diverse role in signaling and biological function. The sphingosine–1–phosphate receptor (S1PR) family has five members that are broadly expressed throughout human tissues and mediate signaling critical to vascular maturation, hair cell formation and repair, T and natural killer – cell trafficking and function. My goal was to examine how naturally occurring genetic variants affected S1PR function and signaling capabilities. For the analysis, I have developed a yeast model system adapting the yeast, Saccharomyces cerevisiae, pheromone signaling pathway to permit a simple and elegant system to perform single receptor analysis. For example, the S1PR1 variant R13G leads to an increase in potency (EC50) of S1P when compared to the wild type receptor. In S1PR2, the R60Q variant, decreases the potency of S1P compared to the wild type receptor. Most interestingly, the S1PR5 variant L318Q had little effect on S1P signaling mediated by Gαi2–chimeric G protein, yet abolished signaling via Gα12-chimeras. To begin to translate these findings, human cells either transiently or stably transduced with S1PR5 and the L318Q variant were studied. Compared to the wild type S1PR5, the L318Q variant caused decreased f-actin accumulation and less stress fiber and filopodia formation when treated with S1P. This region in S1PR5 was predicted to be post–translationally modified with the addition of a palmitoyl group at two cysteine residues at positions 322 and 323. Acyl–biotin exchange experiments confirmed the S1PR5 L318Q variant, unlike the wild type receptor, is unable to be palmitoylated. To test if the cysteine residues were essential for the Gα12 interaction, the C322A and C323A double mutant was created. This receptor replicated the unusual G protein coupling pattern observed in L318Q receptor. I also performed experiments here where the variant receptors were examined for any dysfunction when treated with current and experimental drugs for relapsing remitting multiple sclerosis (RRMS). I discovered minimal effects caused by the variants compared to the signaling via activation of S1P, however I do observe significant differences between the drugs when comparing potency and efficacy. A major finding from those studies is that the experimental drug RPC1074 displays higher efficacy and potency of S1PR1 signaling when compared to the current standard treatment of fingolimod. Examination of the other drugs ponesimod, siponimod, and MT1303p showed similarities in signaling effects when compared to fingolimod. One important finding of this work was discovered when I examined the allelic distribution of the S1PR L318Q polymorphism and found it to be highly enriched in South Asian populations compared to the average population frequency. I also observed an enrichment of another S1PR variant, S1PR1 A11D, in people of African descent. Since S1P metabolism and signaling has been found to be involved in both resistances to and survival from malarial infection, I hypothesize that the L318Q variant by affecting the expression of S1P lyase, an important mediator of S1P metabolism, may affect the response to malarial infections. These ideas are worthy of future experimentation.