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.