Structure-function relationships of two loss-of-function mutations of the thyrotropin receptor gene.
The functional and structural characteristics of two previously described "loss-of-function" mutants of the thyrotropin receptor (TSHR) gene were analyzed by transient transfection in COS cells. Both mutations (Pro162Ala, Ile167Asn) are located in the putative extracellular hormone-binding domain of the receptor. The following parameters were analyzed: expression of native receptor on the cell surface (as measured by binding of labeled thyrotropin [TSH] to intact cells, or flow cytometry of intact cells); total TSHR expression (measured by flow cytometry of permeabilized cells); response to TSH measured as cyclic adenosine monophosphate (cAMP) accumulation. The total cellular expression of both mutant receptors was similar. Cell surface expression of Pro162A1a mutant was reduced about twofold and the EC50 for TSH stimulation was increased twofold. In contrast, the Ile167Asn mutant did not reach the cell surface and the intracellularly expressed mutant protein did not react with a monoclonal antibody (BA8) recognizing only the native TSHR. Based on the current model of the three-dimensional structure of the TSHR, the Pro162Ala substitution maps at the surface of the molecule, while the Ile167Asn mutation affects a residue whose side chain contributes to the hydrophobic core characteristic of proteins harboring leucine repeat motifs. These results are consistent with Ile167Asn causing a gross destabilization of receptor structure incompatible with its normal routing through the intracellular membrane system of the cell.
Costagliola
S
S
Institut de Recherche Interdisciplinaire, Bangkok, Thailand.
Sunthorntepvarakul
T
T
Migeotte
I
I
Van Sande
J
J
Kajava
A M
AM
Refetoff
S
S
Vassart
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D019556:COS Cells; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007930:Leucine; D051379:Mice; D009154:Mutation; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D020449:Repetitive Sequences, Amino Acid; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP; D007930:Leucine