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

Susanne Neumann, G. Krause, S. Chey, Ralf Paschke
2001 Molecular Endocrinology  
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
more » ... 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. (Molecular Endocrinology 15: 1294-1305, 2001) T HE TSH RECEPTOR (TSHR) is a member of the superfamily of G protein-coupled receptors (GPCRs) (1, 2), which also includes the FSH receptor and the LH/CG receptor (LHR) in the subfamily of glycoprotein hormone receptors (3, 4). Sequence alignment of GPCRs has revealed a high degree of homology in their transmembrane domains (TMs), suggesting similarities in transmembrane structural features and signal transduction mechanisms (5). Activation of these receptors by their diverse agonists is associated with crucial conformational changes in the receptor molecule resulting in a movement of transmembrane helices relative to one another and subsequent G protein activation (6). As for most GPCRs, the precise molecular mechanism of the TSHR activation is not known. The understanding of the intramolecular interactions and conformational changes underlying receptor activation is hindered by a lack of information on the three-dimen-sional high-resolution structure of the TSHR. Lowresolution structure data (6 Å) from cryomicroscopy data of frog rhodopsin suggested a tilt of the transmembrane segments. However, they could not provide structural information about individual side chains or even atoms (7, 8) . Very recently the x-ray crystal structure of the bovine rhodopsin with a resolution of 2.8 Å provided new, important insights into side chain orientations of rhodopsin (9). In this model some helical portions of TM2, TM5, TM6, and TM7 are not organized in an ideal ␣-helical backbone structure, which may be due to particular amino acid residues. Noteworthy, in comparison to rhodopsin, glycoprotein hormone receptors have different residues in almost all respective critical positions; thus an identical side chain orientation is very unlikely. Site-directed mutagenesis and the evaluation of its effects on receptor binding and signal transduction provide a valid means of obtaining insights into intramolecular changes of glycoprotein hormone receptors, as well as GPCRs in general, during activation (5). Moreover, molecular modeling is necessary to integrate experimental observations and biophysical and structural data into a mechanistic three-dimensional
doi:10.1210/mend.15.8.0672 pmid:11463854 fatcat:6qh6i6pldrgqpabaigddf637ce