Abstract
Gonadotropin receptors are members of the seven-transmembrane (TM) receptor family. Several point mutations in TM V and VI and the intracellular loop 3 (i3) have been identified in the luteinizing hormone (LH) receptor gene, leading to constitutive activation of the receptor. Because gonadotropin receptors are highly conserved, we mutated the follicle-stimulating hormone (FSH) receptor at the corresponding amino acids. However, the FSH receptor mutants showed minimal increases in basal cAMP production. Taking advantage of this difference between the two receptors, we designed chimeric receptors with or without a point mutation in the i3 to identify the region in the LH receptor important for its constitutive activation. Introduction of the point mutation into chimeric receptors containing only TM V to VI from the LH receptor led to major increases in ligand-independent cAMP production. Furthermore, a chimeric receptor with only TM V and VI derived from the LH receptor can be rendered constitutively active by the mutation in the i3 from the FSH receptor. These results suggest that interactions between TM V and VI of the FSH receptor are essential for maintaining the receptor in the more constrained state, whereas interactions between these domains of the LH receptor are permissive for constitutively activating mutations in the i3.
Highlights
The seven-TM1 G protein-coupled receptors probably represent one of the largest gene family in eukaryotic organisms
The present findings demonstrated that the introduction of single amino acid mutations in the i3 and the seventransmembrane (TM) VI of the human follicle-stimulating hormone (FSH) receptor, unlike similar changes in the luteinizing hormone (LH) receptor (17, 19), did not lead to constitutive receptor activation
Studies using chimeric receptors further indicated that the extracellular region was important for ligand binding but not involved in receptor activation, whereas the TM V to VI region of the LH receptor was essential for constitutive activation
Summary
Hormones and Reagents—Purified hCG (CR129) and FSH (I-3) were supplied by the National Hormone and Pituitary Program, NIDDK, NIH. When cells were 70 – 80% confluent, the medium was replaced with Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.), and transient transfection was performed using up to 12 g of expression vector with or without cDNA inserts by the calcium phosphate precipitation method (29). Forty-eight hours after transfection with increasing amounts of plasmids (0.1–2 g/well), cells were washed once with PBS and incubated for 90 min at 37 °C in the presence of 0.25 mM 3-isobutyl-1-methyl xanthine (Sigma). Ligand Binding Analysis—Purified FSH (I-3) and hCG (CR129) were iodinated by the lactoperoxidase method (33) and characterized by radioligand receptor assay using recombinant human gonadotropin receptors stably expressed in 293 cell line. 200,000 cells/300 l were incubated with increasing amounts of 125I-hCG or 125I-FSH at room temperature for 18 –22 h in the presence or the absence of excess amount of unlabeled hCG (Pregnyl, 100 IU/tube) or unlabeled recombinant human FSH (2.5 IU/tube). CAMP production was normalized based on the number of expressed receptors derived from Scatchard plot analysis
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