An Activation Switch in the Rhodopsin Family of G Protein-coupled Receptors

Abstract

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche؉ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of ϳ14 and ϳ10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of ϳ13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.Genome sequencing projects have identified the G proteincoupled receptor (GPCR) 1 family as one of the largest class of proteins with more than 800 human sequences. These sequences have been classified into five main families, glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin (1). GPCRs are involved in passing chemical signals across the cell membrane. The incoming signal may arrive in the form of neurotransmitters, peptides, divalent cations, proteases, hormones, and sensory stimuli such as photons and gustatory or odorant molecules (2, 3). Atomic level details of a three-dimensional structure of a GPCR are only known for the inactive form of rhodopsin, the light photoreceptor protein of rod cells (4, 5). Rhodopsin is formed by an extracellular N terminus, seven ␣-helices, which cross the cellular membrane (TM 1-TM 7) connected by hydrophilic loops, and a cytoplasmic C terminus containing an ␣-helix (HX 8) parallel to the cell membrane. The overall structure of the helical bundle seems common in the rhodopsin-like GPCR family because of the large number of conserved sequence patterns in these 7 TM segments (6): GN in TM 1, (N/S)LxxxD in TM 2, (D/E)RY in TM 3, CWxP in TM 6, and NPxxY(x) 5,6 F in TM 7 and HX 8, among others. Activation of GPCRs is commonly discussed in terms of the extended ternary complex model, which proposes an equilibrium between inactive and active states (7). Recent advances in the ␤ 2 -adrenergic receptor have shown that ag...

Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers

“…1a, color corresponds to each substitution). Indeed, as reported before, all these point mutations caused an increase in basal cyclic AMP accumulation that, when normalized to the level of expression of each mutant allows sorting them according to their 'specific constitutive activity' 35 (Fig. 1b).…”

“…Concentration-effect curves were fitted with Prism v5.0 and EC 50 calculated. Specific constitutive activity was calculated exactly as described previously 35 , based on the linear relationship existing between basal cAMP accumulation and receptor surface expression.…”

“…To investigate how the protomers communicate within a GpHr homomer, we evaluated the effect of ligand-independent constitutive activity on TSHr hormone binding. To avoid direct influence of mutations on the binding site, thereby introducing potential structural bias, we explored several point mutations known to be constitutively active [33][34][35] and randomly distributed in the receptor sequence (Fig. 1a, color corresponds to each substitution).…”

“…All the constructions were engineered by subcloning available mutant receptors. TSHr mutations were created by subcloning the mutant receptors already constructed in pSVL 33,35 into the AflIII/XbaI or Bsu36I/XbaIdigested pcDNA3.1-SPRT-TSHr cDNA. For LH/CGr, the previously constructed mutant receptors (Rodien and Bonomi, unpublished) were subcloned into the HindIII/BamHI-digested pcDNA3.1-SPRT-LH/CGr cDNA.…”

Glutamate Acts as a Partial Inverse Agonist to Metabotropic Glutamate Receptor with a Single Amino Acid Mutation in the Transmembrane Domain

“…The XPKXY motif has been considered as a key motif equivalent to the well characterized NPXXY motif in family 1 GPCRs (27). In the case of glycoprotein hormone receptor, Thr 6.43 and Asp 6.44 interact mainly with Asn 7.49 in the NPXXY motif to keep the receptor inactive (31,32), and naturally occurring constitutively active mutations at these loci cause genetic disorders such as hyperfunctioning thyroid adenoma and familial male-limited precocious puberty (6). Moreover, the recent crystal structure of constitutively active mutant M257Y 6.40 of rhodopsin showed that elevation of the basal activity originated from the formation of specific interactions among helices III, V, VI, and VII including M257Y…”