A Free Carboxylate Oxygen in the Side Chain of Position 674 in Transmembrane Domain 7 Is Necessary for TSH Receptor Activation

Abstract

A specific H-bonding network formed between the central regions of transmembrane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of transmembrane domain 6. Position D633 in transmembrane domain 6 of the human TSH receptor is the only one in which four different constitutively activating amino acid exchanges have been identified. Further in vitro substitutions led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential interaction partners of R633 in transmembrane domain 3 and/or transmembrane domain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H-bond network that most likely maintains the inactive state of the TSH receptor, we investigated these potential interactions by generating TSH receptor double mutants designed to break up possible H bonds. We excluded S508 in transmembrane domain 3 as a possible interaction partner of R633. In contrast, a partial response to TSH stimulation was rescued in a receptor construct with the double-substitution D633R/N674D. Our results therefore confirm the H bond between position 633 in transmembrane domain 6 and 674 in transmembrane domain 7 suggested by molecular modeling of the inactivating mutation D633R. Moreover, the mutagenesis results, together with a three-dimensional structure model, indicate that for TSH receptor activation and G protein-coupled signaling, at least one free available carboxylate oxygen is required as a hydrogen acceptor atom at position 674 in transmembrane domain 7.

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

“…In this mode of interaction the polar O ␦ atom of Asn-7.49 is free and points toward the intracellular side (see below). This carboxylate oxygen has been suggested as necessary in TSHr activation (35).…”

Interactions between the extracellular domain and the extracellular loops as well as the 6th transmembrane domain are necessary for TSH receptor activation

“…In previous studies, we (36) and others (45) have shown that D633 in the 6th TMD is involved in the H-bond network between the 6th TMD and the 7th TMD and therefore is important for TSHR activation. The substitution of D633 with a positively charged arginine locked the TSHR in a completely inactive state (36).…”

“…The substitution of D633 with a positively charged arginine locked the TSHR in a completely inactive state (36). The introduction of the D633K mutation also strongly reduced TSHR activity (Table 1), most likely also by disturbing the intramolecular H-bond network and lowering the mobility of the 6th and 7th TMD.…”

The Antibodies against the Computationally Designed Mimic of the Glycoprotein Hormone Receptor Transmembrane Domain Provide Insights into Receptor Activation and Suppress the Constitutively Activated Receptor Mutants

“…11C). The interhelical network of hydrogen bond between Leu-457, Asp-578, and Asn-619 has been shown to be highly conserved in all the three GpHR members as shown through the rearrangement of carboxylate oxygen of TSHR N674 (Asn-619 in LHR) with Asp-633 (Asp-578 in LHR), resulting in a switch between the activated and inactivated states (29). Control of such an interhelical molecular switch by modulating the ECLs has already been exploited in designing small molecule agonist for TSHR and LHR (19).…”