Significance of ectodomain cysteine boxes 2 and 3 for the activation mechanism of the thyroid-stimulating hormone receptor.
Recently, we identified constitutively activating mutations at positions Asp-403, Glu-404, and Asn-406 in the third extracellular cysteine box (C-b3) of the thyroid-stimulating hormone receptor. We hypothesized that this region could act as a molecular interface between the extracellular and serpentine domain. In this study we present a model for properties of potential interaction partners for this region. Moreover, we show that Pro-400 and Pro-407 adjacent to this epitope are also important for stabilizing the partially active, basal conformation of the wild-type (WT) thyroid-stimulating hormone receptor. Furthermore, the mutation K291A in the second extracellular cysteine box (C-b2) was identified as a new constitutively activating mutation that releases the basal conformation of the WT receptor like the known tryptic cleavage in its close vicinity. Taken together, we provide an activation scenario at the C-b2/C-b3 unit. Three anchor fragments (anchors I-III) most likely constrain the basal conformation. The three anchor fragments are tightly packed. A disulfide bridge holds the C-b2/C-b3 portions in close positions. Independent of the type of conformational interference such as side chain modifications, tryptic cleavage, or hormone stimulation that act on the constrained C-b2/C-b3 WT conformation, it will always release one of the anchor fragments. Subsequently, this results in a conformational displacement of the C-b2/C-b3 portions relative to each other, inducing receptor activation.
Mueller
Sandra
S
III Medical Department, University of Leipzig, Philipp-Rosenthal-Strasse 27, D-04103 Leipzig, Germany.
Kleinau
Gunnar
G
Jaeschke
Holger
H
Neumann
Susanne
S
Krause
Gerd
G
Paschke
Ralf
R
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000502:physiology*; D014357:Trypsin / Q000737:chemistry
D004220:Disulfides; D000939:Epitopes; D011989:Receptors, Thyrotropin; D014357:Trypsin; D003545:Cysteine