Inhae Ji

The Luteinizing Hormone/Chorionic Gonadotropin Receptor Has Distinct Transmembrane Conductors for cAMP and Inositol Phosphate Signals

The luteinizing hormone/chorionic gonadotropin receptor is a member of the seven-transmembrane receptor family. It is coupled, presumably via Gs and Gq, to two signal pathways involving adenylyl cyclase/cAMP and phospholipase C/inositol phosphate (IP). Little is known about the events prior to G-protein coupling: for example, whether these signals are generated from a single or multiple independent origins and mechanisms, when and where they diverge, and how they are transduced. We report novel observations that the cAMP signal and the IP signal originate and diverge upstream of G-protein coupling. The generation of these two signals independently involves Lys583 in exoloop 3 of the rat receptor. For this study, Lys583 of the receptor was substituted with a panel of amino acids, and mutant receptors were assayed for hormone binding and induction of cAMP, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. No substitutions for Lys583 were permissible for cAMP induction, despite successful surface expression and hormone binding. In contrast, several substitutions were permissible for IP induction. Our results suggest two distinct transmembrane signal conductors for cAMP and inositol phosphate signals and imply particular models of receptor activation not previously suggested.

Kalirin is Under-Expressed in Alzheimer's Disease Hippocampus

To identify genes aberrantly expressed in the brain of individuals with Alzheimers Disease (AD), we analyzed RNA extracts from the hippocampus and cerebellum from 19 AD patients and 15 age- and sex-matched control subjects. Our analysis identified a number of genes that were over-expressed or under-expressed specifically in AD hippocampus. Among these genes, kalirin was the most consistently under-expressed in AD hippocampus, which was verified by semi-quantitative RT-PCR and real time PCR. Kalirin is predominantly expressed in the brain, particularly in the hippocampus, and plays crucial roles in neuronal stability and growth. Our observation is the first to relate kalirin to AD and a human disease. In addition to kalirin, the genes for voltage-gated Ca++ channel gamma subunit 3 and visinin-like protein 1 (a Ca++ sensor protein) were under-expressed, whereas inositol 1,4,5-triphosphate 3-kinase B was over-expressed in AD hippocampus. Collectively, these differential expressions could severely impair calcium homeostasis. Remarkably, these aberrant gene expressions in AD hippocampus were not observed in AD cerebellum. Furthermore, housekeeping genes such as ribosomal protein genes are not affected by AD. These results provide new insights into the biochemistry of AD.

Analyses of ovine corpora lutea for tumor necrosis factor mRNA and bioactivity during prostaglandin-induced luteolysis

It has been suggested that tumor necrosis factor (TNF) participates in the mechanism of regression of the corpus luteum. We measured luteal expression of TNF alpha mRNA and biological activity during prostaglandin-induced luteolysis in sheep. Initiation of functional luteolysis was marked by a sharp decline in concentrations of progesterone in luteal tissue beginning 4 h after administration of luteolysin. Structural regression of corpora lutea was manifested by a reduction in glandular weight at 16 h. A luteal cytotoxic factor with TNF alpha-like bioactivity was isolated after the decrease in tissue progesterone had occurred, but before evidence of luteal resorption. We were unable to detect temporal alterations in TNF alpha mRNA in luteal samples by classical Northern blot or in situ hybridization analyses. These results imply that luteal TNF alpha is derived primarily as a preformed entity from an extraovarian source, such as infiltrating leukocytes. These results raise the possibility that this cytokine might not be involved in the early stages of luteal regression in the ewe, yet could play a secondary role, perhaps in the subsequent opsonization and removal of degenerating cells.

The Amino-terminal Region of the Luteinizing Hormone/Choriogonadotropin Receptor Contacts Both Subunits of Human Choriogonadotropin I. MUTATIONAL ANALYSIS

The luteinizing hormone/choriogonadotropin receptor is a seven-transmembrane receptor. Unlike most seven-transmembrane receptors, it is composed of two halves of equal size, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. The exodomain is exclusively responsible for high affinity hormone binding, whereas receptor activation occurs only in the endodomain. This mutually exclusive physical separation of the two functional domains sets the lutropin receptor and its subfamily of receptors apart from all other seven-transmembrane receptors. The mechanisms of hormone binding and receptor activation also appear to be different from those of other receptors in that binding occurs in at least two steps. However, the precise hormone contact sites in the exodomain are unknown. To determine the hormone/receptor contact sites, we have examined the receptor using progressive truncation from the C terminus, Ala scanning, immunofluorescence microscopy, and antibody binding. Progressive truncation from the C terminus of the receptor indicates several discrete regions that impact hormone binding. These regions are around the boundaries of exons 1–2, 4–5, 6–7, and 9–10. Ala scanning of the Asp17–Arg26 region near the exon 1–2 junction uncovered three alternating residues (Leu20, Cys22, and Gly24) crucial for hormone binding. Ala substitution for any one of these residues abolished hormone binding, although the resulting mutant receptors were successfully expressed on the cell surface. In contrast, Ala substitution for their flanking and intervening residues did not impair hormone binding. These results and the data in the accompanying article (Phang, T., Kundu, G., Hong, S., Ji, I., and Ji, T. (1998)J. Biol. Chem. 273, 13841–13847) indicate that this region directly contacts the hormone and suggest a novel mode of embracing the hormone.

Activation of membrane receptors

Many extracellular messengers interact with discriminate receptors on the cell surface. Some of bound ligands activate receptors whereas others fail to do so. Only activated receptors are capable of generating and transferring signal through the membrane. Recent advances in our understanding of agonist-induced and constitutive receptor activation suggest several molecular mechanisms for receptor activation, signal generation and transmembrane signal transfer.

Roles of Transmembrane Prolines and Proline-induced Kinks of the Lutropin/Choriogonadotropin Receptor

The lutropin/choriogonadotropin receptor is a seven-helix transmembrane (TM) receptor. A unique feature of TM helices is the content of Pro, which generally is absent in α helices of globular proteins. Because Pro disrupts helices and introduces a ∼26° kink, it has been speculated that Pro plays a crucial role in the structure of TM helices, exoloops, and cytoloops of TM receptors. To examine the roles of the five TM Pros of the lutropin/choriogonadotropin receptor, these residues were individually substituted. Mutant receptors were examined for surface expression, hormone binding, and cAMP induction. Surface expression was monitored after introducing the flag epitope into the receptors. Flag epitopes slightly affected cAMP induction but not hormone binding or surface expression of receptors as monitored by immunofluorescence microscopy and 125I-anti-flag antibody. The results indicate that Pro479 in TM 4 and Pro598 in TM 7 play important yet contrasting roles. Pro479 is crucial for hormone binding at the cell surface but not after solubilization of the receptor. This is more likely due to the Pro side chain than the Pro-induced kink. Pro598 is important for surface expression. The kinks of Pro463 of TM 4, Pro562 of TM 6, or Pro591 of TM 7 are not important because the substitution of Phe for these residues did not significantly impact surface expression, hormone binding, and cAMP induction.

Under-expression of Kalirin-7 Increases iNOS Activity in Cultured Cells and Correlates to Elevated iNOS Activity in Alzheimer's Disease Hippocampus1

Recently, it has been reported that Kalirin gene transcripts are under-expressed in AD hippocampal specimens compared to the controls. The Kalirin gene generates a dozen Kalirin isoforms. Kalirin-7 is the predominant protein expressed in the adult brain and plays crucial roles in growth and maintenance of neurons. Yet its role in human diseases is unknown. We report that Kalirin-7 is significantly diminished both at the mRNA and protein levels in the hippocampus specimens from 19 AD patients compared to the specimens from 15 controls. Kalirin-7 associates with iNOS in the hippocampus, and therefore, Kalirin-7 is complexed with iNOS less in AD hippocampus extracts than in control hippocampus extracts. In cultured cells, Kalirin-7 associates with iNOS and down-regulates the enzyme activity. The down-regulation is attributed to the highly conserved 33 amino acid sequence, K(617) -H(649), of the 1,663 amino acids long Kalirin-7. Remarkably, the iNOS activity is considerably higher in the hippocampus specimens from AD patients than the specimens from 15 controls. These observations suggest that the under-expression of Kalirin-7 in AD hippocampus correlates to the elevated iNOS activity.

The Amino-terminal Region of the Luteinizing Hormone/Choriogonadotropin Receptor Contacts Both Subunits of Human Choriogonadotropin II. PHOTOAFFINITY LABELING

The luteinizing hormone/choriogonadotropin receptor, a seven-transmembrane receptor, is composed of two equal halves, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. Unlike most seven-transmembrane receptors, the exodomain alone is responsible for high affinity hormone binding, whereas signal is generated in the endodomain. These physical separations of hormone-binding and receptor activation sites are attributed to unique mechanisms for hormone binding and receptor activation of this receptor and its subfamily members. However, the precise hormone contact sites in the exodomain are unclear. In the preceding article (Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835–13840), a region immediately downstream of the N terminus of the exodomain was shown to be crucial for hormone binding. To test if the region interacts with the hormone, human choriogonadotropin (hCG) was photoaffinity-labeled with a peptide mimic corresponding to Gly18–Tyr36 of the receptor. This peptide mimic specifically photoaffinity-labeled both the α- and β-subunits of hCG. Interestingly, hCGα was preferentially labeled. On the other hand, denatured hCG was not labeled, and a mutant analog of the peptide failed to label hCG. Furthermore, the affinity labeling was UV-dependent and saturable, indicating the specificity of the photoaffinity labeling. Our results indicate that the region of the exodomain interacts with hCG and that the contact points are near both subunits of hCG. Particularly, the alternate residues (Leu20, Cys22, and Gly24) are crucial for hCG binding. In addition, the results underscore the fact that there is a crucial hormone contact site outside of the popularly believed primary hormone-binding site that is composed of Leu-rich repeats and is located in the middle of the exodomain. Our observations are crucial for understanding the molecular mechanism through which the initial high affinity hormone binding leads to receptor activation in the endodomain.

Follicle-stimulating hormone interacts with exoloop 3 of the receptor.

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of ∼330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of580KVPLITVSKAK590 was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH α but not FSH β. This is in contrast to photoaffinity labeling of FSH β by the peptide mimic of the N-terminal region of the receptor. Leu583 and Ile584 are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu583 and Ile584 side chains appear to project in opposite directions. Ile584 appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu583 is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu583 improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile584 and Lys590abolished the cAMP and IP induction. Our results open a logical question whether Leu583, Ile584, and Lys590 interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.

Trans-activation, cis-activation and signal selection of gonadotropin receptors

It has been thought that when a hormone binds to a receptor, the liganded receptor activates itself and generates hormone signals, such as the cAMP signal and the inositol phosphate signal (cis-activation). We describe that a liganded LH receptor or FSH receptor molecule is capable of intermolecularly activating nonliganded receptors (trans-activation). Particularly, intriguing is the possibility that a pair of compound heterozygous mutants, one defective in binding and the other defective in signaling, may cooperate and rescue signaling. Furthermore, trans-activation of the binding deficient receptors examined in our studies generates either the cAMP signal or the IP signal, but not both. Trans-activation and selective signal generation have broad implications on signal generation mechanisms, and suggest new therapeutic approaches.