Transfer of information across membranes is fundamental to the function of all organisms and is primarily initiated by transmembrane receptors. This is an allosteric process and involves conformational coupling between ligand-binding domain and signaling domain of a receptor. This allosteric mechanism of activation is unclear for many receptors. Moreover, for many receptors, how ligand sensitivity is fine-tuned and how disease associated mutations modulate receptor conformation to allosterically affect receptor sensitivity are unknown. The calcium-sensing receptor (CaSR) is a dimeric class C G protein-coupled receptor (GPCR) responsible for maintaining stable extracellular calcium levels in vertebrates. CaSR is more broadly activated by polyamines (spermine and spermidine) and polycations (Ca2+, Mg2+, Sr2+, Ba2+, Gd3+, and La3+), and the sensitivity of CaSR to these ligands is augmented by L-amino acids. At the beginning of this dissertation work in 2016, there were only two published structures of a truncated CaSR construct. These structures provided a basis for understanding the structural rearrangement necessary for receptor activation, but a critical understanding of the individual contributions of polycations and L-amino acids was lacking. Furthermore, the published structures represent a static image and may not represent the dynamic activation process of full length CaSR. Additionally, it has been demonstrated that CaSR from different species can have large differences in ligand sensitivity. In this study, single-molecule FRET (smFRET), computational analysis, and a cell signaling assay is used to answer these questions. I found that CaSR undergoes unique conformational rearrangements compared to other class C GPCRs owing to specific structural features. Moreover, the analysis of disease associated mutations uncovered a large permissiveness in the architecture of the extracellular domain of CaSR with dynamics and not specific receptor topology determining the effect of a mutation. Finally, a structural hub at the dimer interface allosterically controls CaSR activation via focused electrostatic repulsion. Changes in the surface charge distribution of this hub, which is highly variable between organisms, finely tunes CaSR sensitivity. This design is likely a general tuning design for other dimeric receptors. Together, the studies in this dissertation highlight the importance of probing the conformational change of proteins in real time.

Creator

• Schamber, Michael Robert

DOI

• https://doi.org/10.21985/n2-xpkt-3z92

Subject

• Bioinformatics
• Biochemistry
• Biophysics

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:16268
• etdadmin_upload_929368

Keyword

• GPCR
• CaSR
• smFRET

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright