From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

Abstract

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-593 5.50 ) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular ␣-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by nonpolar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences. G-protein-coupled receptors (GPCRs)3 are mediators between extracellular stimuli and intracellular signaling cascades. Knowledge concerning the mechanisms of receptor and G-protein activation has grown exponentially in the past few decades (1-6). GPCRs are anchored in the membrane, and different parts of GPCRs are responsible for specific intra-as well as intermolecular functions during a sequential signal transduction process. Despite individual and selective properties of particular receptors in each of these steps, general aspects are common for family A GPCRs. A "global toggle-switching" mechanism is proposed based on a vast amount of experimental and structural data (7) whereby a vertical see-saw movement of transmembrane helix (TMH) 6 occurs around a pivot. Consequently, spatial movement of particular TMHs relative to each other characterizes receptor activation; this occurs to the greatest extent between TMHs 5, 6, and 7, respectively (8, 9). The conformations and structural rearrangements are supported by amino acids acting as "microswitches" (10). Some of these residues are well known for family A GPCRs such as the highly conserved NPXXY (TMH7) and DRY (TMH3) motifs. Different GPCR conformations are causally related to different signaling activity states (3, 10, 11). Furthermore, differences between activated conformations depend on particular interacting ligands, and they are probably involved in determination of G-protein subtype preferences (12-17). The previously published GPCR structures of inactive receptor conformations (for reviews, see Refs. 15, 18, and 19) compared with the recently published active conformation of opsin and ␤ 2 -adrenergic rece...

Probing the Interaction of SR141716A with the CB1 Receptor

“…In support, it was well established that TM2/TM3/TM7 stabilization was important for the inactive state of the family A GPCRs (40,41 (31).…”

“…Thus, the binding of the C3-piperidinyl moiety of SR141716A stabilized TM2, and the binding of the C5-aromatic ring moiety of SR141716A stabilized TM5. These stabilizing interactions appeared critical for positioning the ligand's N1-aromatic ring moiety to secure the Trp-356 6.48 toggle switch from activation, contributing to the TM2/TM3/TM7 stabilization important for maintaining the inactive state of the receptor (40,41). Furthermore, these interactions prohibit a TM6 rigid-body movement required for receptor activation (50,51), as shown in Fig.…”

Loss-of-Function Variants in a Hungarian Cohort Reveal Structural Insights on TSH Receptor Maturation and Signaling

“…This is also true for other determined inactive GPCR crystal structures such as the ␤1 (pdb entry code 3ZPR) and ␤2 (pdb entry code 2RH1) adrenoceptors or the -opioid receptor (pdb entry code 4DJH) (structures not shown). Third, N432 1.50 is spatially close, but is not directly connected with the H-bond network between conserved residues in helices 2, 3, and 7 (D460 [H2], S508 [H3], N670 [H7], N674 [H7]), which stabilizes the inactive conformation at the central transmembrane core (19,20). In conclusion, our model suggests that N432 1.50 is a separate, highly conserved key player in maintaining the architecture of the TSHR by stabilizing the interface between H1 and H7.…”

New Pathogenic Thyrotropin Receptor Mutations Decipher Differentiated Activity Switching at a Conserved Helix 6 Motif of Family A GPCR

“…TSHR homology models were generated due to a protocol described in a recent study (19). The inactive TSHR conformation (white ribbon backbone) is distinguished from the activated conformation (beige backbone, left panel) by a relative spatial shift of particular TMH to each other.…”