The C-terminal third of the human luteinizing hormone (LH) receptor is important for inositol phosphate release: analysis using chimeric human LH/follicle-stimulating hormone receptors.
Gonadotropin and TSH receptors represent a subgroup of seven transmembrane-spanning, G protein-coupled receptors with a large extracellular ligand-binding region. After ligand binding to their receptors, the majority of actions of gonadotropins and TSH are believed to be mediated by the cAMP-protein kinase A pathway. Although formation of inositol phosphates (IP) has been reported after stimulation of rodent gonadotropin receptors, activation of phospholipase C after ligand binding of human LH or FSH receptors has not been investigated. Human gonadotropin receptors were transiently expressed in 293 cells, and the agonist-induced stimulation of IP formation was measured. The LH receptor responded to a saturating dose of human CG (hCG) with a 5.2-fold increase of IPs whereas the FSH receptor responded to a saturating dose of FSH with only a 50% increase. On the basis of these differences and in view of the homologous nature of the two gonadotropin receptors, chimeric receptors were constructed using domain transfer to identify the regions in the human LH receptor important for phosphatidylinositol hydrolysis. Chimeric receptors containing the entire extracellular region of the FSH receptor and the seven transmembrane region plus the cytoplasmic tail of the LH receptor responded to FSH treatment with a 4.7-fold increase in IP accumulation. In contrast, the chimeric receptor with the extracellular region of the LH receptor and the TM region plus the cytoplasmic tail of the FSH receptor responded minimally (50%) to hCG treatment. When the C-terminal third (from TM V to the cytoplasmic tail) of the FSH receptor was replaced with the LH receptor sequence, the chimeric receptor still responded to FSH treatment with a large (6.2-fold) increase in IP release, similar to that of the wild type LH receptor (to hCG), suggesting that C-terminal third of the human LH receptor confers IP signaling ability. This functional domain was further divided into two areas, namely TM V to TM VI and TM VII to the cytoplasmic tail. The chimeric receptors F(I-IV)L(V-VI)F(VII-C)R and F(I-VI)L-VII-C)R, in which these two regions of the FSH receptor were replaced by the corresponding sequences of the LH receptor, responded to FSH treatment with partial increases in phosphatidylinositol hydrolysis (2.0- and 3.7-fold, respectively). Furthermore, when TM VII and the cytoplasmic tail of the LH receptor were replaced with the corresponding sequence of the FSH receptor, this chimeric receptor showed a diminished (2.0-fold) response to hCG in IP release. For all the chimeric receptor constructs analyzed, overall expression, equilibrium binding constants, and adenyl cyclase activation were not altered. Thus, unlike studies using chimeric muscarinic and dopaminergic receptors in which the second and third intracellular loops were found to be important for IP signaling, the entire C-terminal third of the human LH receptor is important for IP release. Future analysis using the chimeric receptor approach should provide new information on the structure-function relationship of gonadotropin, TSH, and other seven transmembrane-spanning receptors.
Hirsch
B
B
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317, USA.
Kudo
M
M
Naro
F
F
Conti
M
M
Hsueh
A J
AJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000378:metabolism; D011962:Receptors, FSH / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011974:Receptors, LH / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity
D006063:Chorionic Gonadotropin; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D011962:Receptors, FSH; D011974:Receptors, LH; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP