Abstract
G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small size among GPCRs, is amenable to molecular biological approaches and to computer modeling. These techniques and interspecies comparisons are used to identify structural features that are important for both intracellular trafficking and GnRHR activation yet distinguish between these processes. Our model features two salt (Arg38-Asp98 and Glu90-Lys121) and two disulfide (Cys14-Cys200 and Cys114-Cys196) bridges, all of which are required for the human GnRHR to traffic to the plasma membrane. This study reveals that both constitutive and ligand-induced activation are associated with a “coincidence detector” that occurs when an agonist binds. The observed constitutive activation of receptors lacking Glu90-Lys121, but not Arg38-Asp98 ionic bridge, suggests that the role of the former connection is holding the receptor in the inactive conformation. Both the aromatic ring and hydroxyl group of Tyr284 and the hydrogen bonding of Ser217 are important for efficient receptor activation. Our modeling results, supported by the observed influence of Lys191 from extracellular loop 2 (EL2) and a four-residue motif surrounding this loop on ligand binding and receptor activation, suggest that the positioning of EL2 within the seven-α-helical bundle regulates receptor stability, proper trafficking, and function.
Footnotes
This work was supported in part by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK85040] (to P.M.C.); the National Institutes of Health National Center for Research Resources [Grants RR030229, RR000163] (to P.M.C.); the National Institutes of Health National Institute on Drug Abuse [Grant DA003910] (to H.I.M.); and by the National Science Foundation [Grant 0849713] (Division of Biological Infrastructure) (to I.D.P.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.180869.
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ABBREVIATIONS:
- GnRH
- gonadotropin-releasing hormone
- GnRHR
- gonadotropin-releasing hormone receptor
- hGnRHR
- human GnRHR
- QCS
- quality control system
- ER
- endoplasmic reticulum
- GPCR
- G protein-coupled receptors
- PM
- plasma membrane
- TM
- transmembrane segment
- CA
- constitutive activity
- WT
- wild type
- EL
- extracellular loop
- DMEM
- Dulbecco's modified Eagle's medium
- BSA
- bovine serum albumin
- IP
- inositol phosphate.
- Received March 1, 2011.
- Accepted April 27, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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