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Structural mechanisms of class B GPCR signaling: the case of the parathyroid hormone receptor

Clark, Lisa / J (2020) Structural mechanisms of class B GPCR signaling: the case of the parathyroid hormone receptor. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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The parathyroid hormone (PTH) type 1 receptor (PTHR), a class B G protein-coupled receptor (GPCR), is the key regulator of bone turnover and mineral ion homeostasis. A thorough understanding of the structural mechanisms of PTHR function is necessary to develop therapeutics for bone and mineral disorders (e.g., osteoporosis). To further this aim, we used an interdisciplinary approach, which included cryo-EM, NMR spectroscopy, and MD simulations to investigate several steps in the PTHR signaling pathway: ligand binding, receptor activation, coupling to effector proteins, and termination of signaling.

We solved high-resolution cryo-EM structures of PTHR in complex with the stimulatory G protein (Gs) for adenylyl cyclases and a long-acting PTH analog (LA-PTH). These structures provide insights into the mechanism of PTHR activation. We also sought determine how LA-PTH triggers prolonged sustained cAMP signaling in endosomes compared to PTH. In MD simulations, the N-terminal part of LA-PTH makes more contacts with PTHR in comparison to PTH. Therefore, LA-PTH remains bound to active receptor despite endosomal acidification, which destabilizes interactions between PTHR and the C-terminal part of the ligand. Next, we probed the mechanism of PTH binding to PTHR. Our results show that initial binding of the PTH C-terminal part to PTHRECD during the first step reduces the mobility of the PTH N-terminal part, which primes the N-terminal part to interact with the receptor transmembrane domain during the second step.

An allosteric link between PTHR interactions with the PTH N-terminal part and receptor coupling to intracellular effectors was demonstrated through the investigation of two PTH mutations, His9->Ala9 (PTHH9A) Leu7 L->D (PTH7d), which inhibit β-arrestin coupling through distinct mechanisms, leading to transient and sustained cAMP production at the plasma membrane, respectively. PTHH9A promotes an inward conformation of receptor intracellular loop 3 to prevent interactions with β-arrestin’s finger loop. PTH7d enhances a receptor polar network stabilizing an outward kink of the intracellular part of transmembrane helix 6 (TM6), which prevents the inward TM6 movement necessary for β-arrestin coupling. In total, our results likely extend to other class B GPCRs and provide a structural framework for drug design targeting bone and mineral disorders.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Clark, Lisa / Jljc32@pitt.eduljc320000-0003-0484-4596
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorVilardaga, Jean-Pierrejpv@pitt.edujpv0000-0002-1217-1435
Committee ChairConway, James / FJames.Conway@pitt.edujxc1000000-0002-6581-4748
Committee MemberRomero, Guillermoggr@pitt.eduggr
Committee MemberZhang, Chengchengzh@pitt.educhengzh0000-0001-9042-4007
Date: 29 December 2020
Date Type: Publication
Defense Date: 2 October 2020
Approval Date: 29 December 2020
Submission Date: 28 October 2020
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 221
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Pharmacology and Chemical Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: parathyroid hormone receptor, structure, allostery, signaling
Date Deposited: 29 Dec 2020 20:50
Last Modified: 29 Dec 2020 20:50


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