A disulfide bonding interaction role for cysteines in the extracellular domain of the thyrotropin-releasing hormone receptor.
The roles of disulfide and sulfhydryl groups in the specific binding of TRH to its receptor have been examined. In all TRH receptors (TRH-Rs) isolated from different species so far, there are only two extracellular cysteine residues (Cys98 in the extracellular loop between transmembrane helices 2 and 3 and Cys179 in the extracellular loop between transmembrane helices 4 and 5) that are in positions homologous to cysteine residues in other G protein-coupled receptors. Another Cys (Cys100) is located in close proximity to Cys98 at the interface between the first extracellular loop and third transmembrane domain. To assess the role of these TRH-R Cys residues in disulfide bonding interactions, they were mutated to either Ser or Ala. Six mutant receptors (Cys98Ser, Cys98Ala, Cys179Ser, Cys179Ala, Cys100Ser, and Cys100Ala) were expressed in COS-1 cells and tested for their ability to bind TRH and to activate total inositol phosphate (IP) formation. TRH-R mutants Cys100Ser and Cys100Ala showed TRH binding affinities and IP activation similar to the wild-type (WT). In contrast, mutants Cys98Ser, Cys98Ala, Cys179Ser, and Cys179Ala showed no high affinity TRH binding. The potencies of Cys98Ala and Cysl79Ala as measured by IP stimulation were decreased by four orders of magnitude when compared with WT. Cys98Ser potency decreased by five orders of magnitude, whereas Cys179Ser showed no IP production. Northern blotting confirmed expression of all the mutant TRH-Rs at the messenger RNA (mRNA) level. An epitope tag derived from the Haemophilus influenza hemagglutinin protein was incorporated at the NH2 termini of the TRH-R WT and TRH-R Cys mutants to allow the independent assessment of cell surface expression of receptor protein. TRH-R mutants that failed to show receptor binding (Cys98Ser, Cys98Ala, Cys179Ala) showed WT levels of cell surface receptor expression, indicating that loss of receptor binding in these mutants is not attributable to loss of receptor expression. In contrast, cell surface expression of Cysl79Ser, which showed no ligand induced IP stimulation, could not be detected. Dithiothreitol, a disulfide bond reducing agent, and p-chloromercuribenzoic acid (p-CMB), a sulfhydryl blocking compound, reduced specific TRH binding in a dose-dependent manner. The inhibition of binding by dithiothreitol implies that the integrity of a disulfide bond is important for TRH binding to its receptor. The dramatic inhibition of TRH binding by p-CMB indicates that free sulfhydryl groups are also associated with the binding of the ligand to its receptor. This study presents evidence that a disulfide bond exists between Cys98 and Cys179 which is essential for maintaining the receptor in the correct conformation for ligand binding. Cys100 is not thought to have a disulfide bonding interaction role. Results obtained after chemical modification have shown that free sulfhydryl groups within the TRH-R may also have a role in ligand interactions.
Cook
J V
JV
Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, Scotland, United Kingdom.
McGregor
A
A
Lee
T
T
Milligan
G
G
Eidne
K A
KA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003545:Cysteine; D017931:DNA Primers; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010903:Pituitary Gland, Anterior / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D017931:DNA Primers; D004220:Disulfides; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D013973:Thyrotropin-Releasing Hormone; D019204:GTP-Binding Proteins; D003545:Cysteine; D004229:Dithiothreitol