Molecular and Pharmacological Analysis of the Chemokine Receptor CCR6
Julian, Bina.
2017
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Abstract: This thesis
advances the molecular understanding of Cys-Cys Chemokine Receptor 6 (CCR6) through
mutagenesis studies and the development of CCR6-based tools that measure and modulate
receptor function. CCR6 is a G protein-coupled receptor (GPCR) whose signaling
influences the behavior of a wide range of cells involved in the inflammatory response.
CCR6 has gained attention for its ... read moreexpression on the Th17 cell type, a proinflammatory T
cell associated with an ever-growing list of chronic inflammatory diseases.
Surprisingly, little is understood about the underlying molecular pharmacology that
influences the GPCR functions of CCR6. To understand the Gαi protein-mediated
signaling of the WT receptor, I utilized well-established in vitro luciferase reporter
assays in combination with the HEK293 cell model. This assay allowed us to observe the
constitutive activity of the WT CCR6 receptor and measure a subnanomolar potency
(0.73nM) of its cognate ligand Cys-Cys Chemokine Ligand 20 (CCL20). Furthermore, this
assay enabled us to compare and contrast the pharmacological properties of naturally
occurring and engineered mutations in CCR6. To understand the susceptibility of the CCR6
protein sequence to activating mutations, I performed mutagenesis studies across three
conserved regions and the C-terminus of CCR6, which identified 12 amino acids
susceptible to activating mutations. Notably, two mutant receptors V255A6.36 and
I256A6.37 exhibited triple the basal activity of the WT receptor while maintaining
comparable ligand potencies. Constitutively active mutants with these properties are
useful tools for drug screening and characterization, therefore I utilized these
receptors to characterize two small molecules. Tests found both compounds to be CCR6
inhibitors, with one exhibiting inverse agonists properties. GWAS studies have linked
CCR6 non-coding polymorphisms with several chronic inflammatory diseases including (e.g.
Crohn's Disease, Rheumatoid Arthritis), however naturally occurring missense mutations
within CCR6 have yet to be characterized. I characterized the five most frequent CCR6
variants (A89T, A150V, R155W, G345S, A369V) and found them all to be loss-of-function,
with A150V and R155W exhibiting a pronounced loss of basal activity and A89T exhibiting
a decreased surface expression. To understand if mutations within CCR6 affect the
ability to recruit beta arrestin, I utilized the bioluminescence resonance energy
transfer (BRET) technique. A set including the five loss-of-function missense variants
and three constitutively active mutants (V255A, I256A, T98P) was evaluated against the
WT receptor. All five naturally occurring variants did not perturb beta arrestin 2
mobilization. However, mutant receptor T98P exhibited constitutive beta arrestin
recruitment, while mutant receptor I256A showed a CCL20-induced increase in
mobilization. To study interactions between CCR6 and CCL20, I applied a membrane
tethered ligand (MTL) strategy that has been used by the Kopin laboratory to probe the
importance of peptide domains and explore GPCR-ligand interactions. Generation and
subsequent mutagenesis of tethered CCL20 highlighted the importance of the ligand N- and
C-terminus of CCL20 in receptor activation. An improved understanding of CCR6-related
physiology and pathophysiology is needed for the design of safe and effective
therapeutic interventions. In an effort to probe underlying molecular and
pharmacological properties that influence CCR6 signaling, this thesis contributes in
vitro tools to expedite CCR6 drug development and loss-of-function variants that deserve
follow up as candidate etiologic factors in selected inflammatory
diseases.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Pharmacology & Experimental Therapeutics.
Advisors: Alan Kopin, and Theoharis Theoharides.
Committee: Martin Beinborn, John Castellot, and Tracy Handel.
Keywords: Pharmacology, Immunology, and Molecular biology.read less - ID:
- 70795m454
- Component ID:
- tufts:20660
- To Cite:
- TARC Citation Guide EndNote
