Abstract
A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity.
Highlights
The glycoprotein hormone receptors (GPHRs)2 LHCGR, FSHR, and thyroid-stimulating hormone receptor (TSHR) constitute a subfamily of the 7TMRs
A structural model of the serpentine domain of TSHR based on the rhodopsin structure [21] orientates the ECL2 between the TMHs, where Ile-568 is located at the tip of ECL2 and is embedded in an environment of hydrophobic residues provided by TMH1, -2, -6, and -7
The ECL2 of the LHCGR has been reported to be involved in hormone binding [30, 37] and to be essential in signal transmission processes [38]
Summary
Site-directed Mutagenesis—The TSHR mutants were constructed by PCR mutagenesis using the human TSHR plasmid TSHR-pSVL as template as described previously [22] PCR fragments were digested with BspTI and Eco91I (MBI Fermentas, Vilnius, Lithuania). Cell Culture and Transient Expression of Mutant TSHRs— COS-7 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen, Paisley, UK) at 37 °C in a humidified 5% CO2 incubator. Cells were washed once with phosphate-buffered saline containing 0.1% bovine serum albumin and 0.1% NaN3 and incubated at 4 °C for 1 h with a 1:200 dilution of a mouse anti-human TSHR antibody (2C11, 10 mg/liter, Serotec Ltd., Oxford, UK) in the same buffer. Forty-eight hours after transfection, cells were preincubated with serum-free Dulbecco’s modified Eagle’s medium containing 1 mM 3-isobutyl-1methylxanthine (Sigma) for 20 min at 37 °C in a humidified 5% CO2 incubator. For cAMP assays, 48 h after transfection, cells were incubated with serum-free Dulbecco’s modified Eagle’s medium containing 1 mM 3-isobutyl-1-methylxanthine (Sigma) for 1 h. The sheet-like fold of ECL2 and its general localization between the transmembrane helices were kept as in rhodopsin based on rhodopsin structure-consistent results for different accessibility of two CC chemokine receptor 5 (CCR5) antibodies, each specific for the two different -strand epitopes of ECL2 of CCR5 (26 –28)
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