Dynamics of m6A RNA Methylome on the Hallmarks of Hepatocellular Carcinoma.

Epidemiological data consistently rank hepatocellular carcinoma (HCC) as one of the leading causes of cancer-related deaths worldwide, often posing severe economic burden on health care. While the molecular etiopathogenesis associated with genetic and epigenetic modifications has been extensively explored, the biological influence of the emerging field of epitranscriptomics and its associated aberrant RNA modifications on tumorigenesis is a largely unexplored territory with immense potential for discovering new therapeutic approaches. In particular, the underlying cellular mechanisms of different hallmarks of hepatocarcinogenesis that are governed by the complex dynamics of m6A RNA methylation demand further investigation. In this review, we reveal the up-to-date knowledge on the mechanistic and functional link between m6A RNA methylation and pathogenesis of HCC.

Autophagy Induced by Simian Retrovirus Infection Controls Viral Replication and Apoptosis of Jurkat T Lymphocytes.

Autophagy and apoptosis are two important evolutionarily conserved host defense mechanisms against viral invasion and pathogenesis. However, the association between the two pathways during the viral infection of T lymphocytes remains to be elucidated. Simian type D retrovirus (SRV) is an etiological agent of fatal simian acquired immunodeficiency syndrome (SAIDS), which can display disease features that are similar to acquired immunodeficiency syndrome in humans. In this study, we demonstrate that infection with SRV-8, a newly isolated subtype of SRV, triggered both autophagic and apoptotic pathways in Jurkat T lymphocytes. Following infection with SRV-8, the autophagic proteins LC3 and p62/SQSTM1 interacted with procaspase-8, which might be responsible for the activation of the caspase-8/-3 cascade and apoptosis in SRV-8-infected Jurkat cells. Our findings indicate that autophagic responses to SRV infection of T lymphocytes promote the apoptosis of T lymphocytes, which, in turn, might be a potential pathogenetic mechanism for the loss of T lymphocytes during SRV infection.

Acetyl-macrocalin B suppresses tumor growth in esophageal squamous cell carcinoma and exhibits synergistic anti-cancer effects with the Chk1/2 inhibitor AZD7762.

Natural products derived from herbal medicines have become a major focus of anti-cancer drug discovery studies. Acetyl-macrocalin B (A-macB) is an ent-diterpenoid isolated from Isodon silvatica. This study aimed to examine the effect and molecular action of A-macB in esophageal squamous cell carcinoma (ESCC) and explore possible drug synergistic modalities. A-macB induced cellular reactive oxygen species (ROS) generation, initiated the p38 mitogen-activated protein kinase (MAPK) signaling pathway, and triggered the caspase-9-dependent apoptosis cascade in ESCC cells. The ROS scavenger N-acetylcysteine (NAC) and the specific p38 inhibitor SB203580 reversed the effects of A-macB on the p38 network and thus rescued ESCC cells from apoptosis. The cellular ROS increase was at least partially due to the suppression of glutathione-S-transferase P1 (GSTP1) by A-macB. A-macB also upregulated the Chk1/Chk2-Cdc25C/Cdc2/Cyclin B1 axis to induce G2/M phase arrest. The cell growth inhibition induced by A-macB was further enhanced by AZD7762, a specific Chk1/Chk2 inhibitor, with a combination index (CI) of <1. Moreover, A-macB efficiently suppressed xenograft growth without inducing significant toxicity, and AZD7762 potentiated the effects of A-macB in the suppression of tumor growth in vivo. Taken together, A-macB is a promising lead compound for ESCC and exerts synergistic anti-cancer effects with AZD7762.

Topological Characterization of Human and Mouse m5C Epitranscriptome Revealed by Bisulfite Sequencing.

BACKGROUND: Compared with the well-studied 5-methylcytosine (m5C) in DNA, the role and topology of epitranscriptome m5C remain insufficiently characterized. RESULTS: Through analyzing transcriptome-wide m5C distribution in human and mouse, we show that the m5C modification is significantly enriched at 5' untranslated regions (5'UTRs) of mRNA in human and mouse. With a comparative analysis of the mRNA and DNA methylome, we demonstrate that, like DNA methylation, transcriptome m5C methylation exhibits a strong clustering effect. Surprisingly, an inverse correlation between mRNA and DNA m5C methylation is observed at CpG sites. Further analysis reveals that RNA m5C methylation level is positively correlated with both RNA expression and RNA half-life. We also observed that the methylation level of mitochondrial RNAs is significantly higher than RNAs transcribed from the nuclear genome. CONCLUSIONS: This study provides an in-depth topological characterization of transcriptome-wide m5C modification by associating RNA m5C methylation patterns with transcriptional expression, DNA methylations, RNA stabilities, and mitochondrial genome.

Functional examination of novel kisspeptin phosphinic peptides.

Kisspeptins acting on their cognate G protein-coupled receptor, kisspeptin receptor, play important roles in the suppression of cancer cell metastasis and regulation of the reproductive system, and therefore are important for therapeutic intervention. All native functional human kisspeptins (kisspeptin-54, kisspsptin-14 and kisspeptin-13) share the 10 amino acids of kisspeptin-10 at their C-terminus (45-54). However, they are inactivated rapidly by matrix metalloproteinases (MMPs) through the cleavage of the peptide bond between glycine51 and leucine52, which limits their clinical applications. Development of MMP-resistant analogues of kisspeptins may provide better therapeutic outputs. In the present study, two kisspeptin phosphinic peptides were designed and synthesized, and their ability to induce phosphorylation of ERK1/2 through kisspeptin receptor and their inhibition on MMP-2 and MMP-9 whose activity correlates with cancer metastasis were assessed. The results showed that one analogue, phosphinic kisspeptin R isomer (PKPR), exhibited kisspeptin receptor-agonistic activity and also inhibitory activity on MMP-2, indicating that PKPR may serve as a lead for the further development of kisspeptin analogues for therapeutic purpose.

Acetyl-macrocalin B, an ent-kaurane diterpenoid, initiates apoptosis through the ROS-p38-caspase 9-dependent pathway and induces G2/M phase arrest via the Chk1/2-Cdc25C-Cdc2/cyclin B axis in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, and novel effective drugs against NSCLC are urgently needed. Isodon species are rich in ent-kaurane diterpenoids that have been reported to have antitumor bioactivity. Acetyl-macrocalin B (A-macB) is a novel ent-kaurane diterpenoid isolated from Isodon silvatica, and its antitumor efficacy against NSCLC and the underlying mechanisms were scrutinized in depth. The viability of cells treated with A-macB was detected by CCK-8 and colony formation assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mechanisms were investigated by detecting ROS and performing western blotting and verification experiments with specific inhibitors. The in vivo effect of A-macB was explored in a nude mouse xenograft model. A-macB effectively inhibited H1299 and A549 cell viability, triggered apoptosis and delayed cells in the G2/M phase. A-macB induced cellular ROS production and then activated the p38 MAPK-mediated, caspase 9-dependent apoptotic pathway. Both the ROS scavenger NAC and the specific p38 inhibitor SB203580 inactivated the function of p38 induced by A-macB, thus preventing cells from apoptosis. A-macB activated the Chk1/2-Cdc25C-Cdc2/cyclin B1 axis to induce G2/M phase arrest. AZD7762 abrogated the function of Chk1/2, abolished the G2/M delay and enhanced the cytotoxicity of A-macB. Moreover, A-macB efficiently suppressed tumor growth in a mouse xenograft model without noticeable toxicity to normal tissues. Having both efficacy and relative safety, A-macB is a potential lead compound that is worthy of further exploration for development as an anticancer agent.

Impaired P2X signalling pathways in renal microvascular myocytes in genetic hypertension.

AIMS: P2X receptors (P2XRs) mediate sympathetic control and autoregulation of renal circulation triggering preglomerular vasoconstriction, which protects glomeruli from elevated pressures. Although previous studies established a casual link between glomerular susceptibility to hypertensive injury and decreased preglomerular vascular reactivity to P2XR activation, the mechanisms of attenuation of the P2XR signalling in hypertension remained unknown. We aimed to analyse molecular mechanisms of the impairment of P2XR signalling in renal vascular smooth muscle cells (RVSMCs) in genetic hypertension. METHODS AND RESULTS: We compared the expression of pertinent genes and P2XR-linked Ca(2+) entry and Ca(2+) release mechanisms in RVSMCs of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats. We found that, in SHR RVSMCs, P2XR-linked Ca(2+) entry and Ca(2+) release from the sarcoplasmic reticulum (SR) are both significantly reduced. The former is due to down-regulation of the P2X1 subunit. The latter is caused by a decrease of the SR Ca(2+) load. The SR Ca(2+) load reduction is caused by attenuated Ca(2+) uptake via down-regulated sarco-/endoplasmic reticulum Ca(2+)-ATPase 2b and elevated Ca(2+) leak from the SR via ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors. Spontaneous activity of these Ca(2+)-release channels is augmented due to up-regulation of RyR type 2 and elevated IP3 production by up-regulated phospholipase C-ß1. CONCLUSIONS: Our study unravels the cellular and molecular mechanisms of attenuation of P2XR-mediated preglomerular vasoconstriction that elevates glomerular susceptibility to harmful hypertensive pressures. This provides an important impetus towards understanding of the pathology of hypertensive renal injury.

The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important role in co-ordinating its activities.

PABP1 [poly(A)-binding protein 1] is a central regulator of mRNA translation and stability and is required for miRNA (microRNA)-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature; however, it is unclear how its different activities are co-ordinated, since many partners interact via overlapping binding sites. In the present study, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations. The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells. Two lysine residues were differentially acetylated or methylated, revealing that PABP1 may be the first example of a cytoplasmic protein utilizing a 'methylation/acetylation switch'. Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection.

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Helix 8 of the M1 muscarinic acetylcholine receptor: scanning mutagenesis delineates a G protein recognition site.

We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M(1) muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein α subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.

A crucial role for Galphaq/11, but not Galphai/o or Galphas, in gonadotropin-releasing hormone receptor-mediated cell growth inhibition.

GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to Galpha(q/11), Galpha(i/o), and Galpha(s), their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in Galpha(q/11)-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to Galpha(i) or chimeric Galpha(qi5), transfection of Galpha(q) cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [35S]GTPgammaS binding assays confirmed that the GnRH receptor was unable to directly couple to Galpha(i) but could directly interact with Galpha(q/11). Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing Galpha(q), and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to Galpha(q/11), but not to Galpha(i/o) or Galpha(s), and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.

Identification of Tyr(290(6.58)) of the human gonadotropin-releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: importance for high-affinity binding and receptor activation.

Molecular modeling showed interactions of Tyr (290(6.58)) in transmembrane domain 6 of the GnRH receptor with Tyr (5) of GnRH I, and His (5) of GnRH II. The wild-type receptor exhibited high affinity for [Phe (5)]GnRH I and [Tyr (5)]GnRH II, but 127- and 177-fold decreased affinity for [Ala (5)]GnRH I and [Ala (5)]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe (5)]GnRH I, GnRH II and [Tyr (5)]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr (290(6.58)) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr (290(6.58)) to Gln increased affinity for Tyr (5)-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr (5) of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr (290(6.58)) side chain with Tyr (5) of GnRH I and His (5) of GnRH II. Tyr (290(6.58)) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.

The synthesis of MP-CDCA conjugates and dissolution kinetics of model cholesterol gallstones.

The comb-like copolymers of polycarboxylic acid were synthesized and then reacted with chenodeoxycholic acid (CDCA) to obtain a series of conjugates, MPn-CDCA, where n is the number of the groups of oxyethylene in each graft chain. This was confirmed by infrared spectroscopy and thin-layer chromatography. We investigated the effects of dissolving model cholesterol gallstones with the MPn-CDCA conjugates in phosphate-buffered saline at pH 7.4. The dissolution rates of CDCA, MP40-CDCA, MP30-CDCA, MP20-CDCA and MP10-CDCA were 5.33, 5.717, 17.59, 6.868 and 9.615x10(-7)kgm(-2)s(-1), micellar solubilities were 0.2431, 3.095, 12.972, 5.248 and 5.790kgm(-3) and total resistances were 5.33, 5.717, 17.59, 6.868 and 9.615x10(-7)kgm(-2)s(-1), respectively. These studies suggested that the interfacial resistance was the dominant rate-determining factor in dissolving model cholesterol gallstones. Model cholesterol gallstones could be more effectively dissolved by increasing the steric interactive potential energy of side chains and ensuring that the hydrophilic-lipophilic properties of MP-CDCA are within an appropriate range. The micellar dissolution rates of model cholesterol gallstones by MP20-CDCA were significantly faster than by the other conjugates.

Gonadotropin-releasing hormone analog structural determinants of selectivity for inhibition of cell growth: support for the concept of ligand-induced selective signaling.

GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His(5), Trp(7), Tyr(8)) were introduced singly or in pairs into GnRH I. Tyr(5) replacement by His(5) produced the highest increase in the antiproliferative potency of GnRH I. Tyr(8) substitution of Arg(8) produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys(6) and D-Arg(6)) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg(8) of GnRH I makes contact with Asp(302), whereas Tyr(8) of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.

Antiproliferative effects of GnRH agonists: prospects and problems for cancer therapy.

Gonadotropin-releasing hormone (GnRH) receptor activation has been demonstrated to inhibit cell proliferation in vitro and in vivo. These effects are dependent on the degree of receptor expression and the intracellular signaling protein milieu. The physiological and pathophysiological relevance is largely undefined, and its potential for exploitation in the treatment of specific malignancies is the subject of ongoing investigations. GnRH receptors are expressed in embryonic, juvenile and adult tissues, including brain, pituitary, gonads, accessory reproductive organs and placenta. The levels of receptor expression vary, from high in pituitary gonadotropes to low in peripheral tissues, although quantification of functional receptor protein has been determined in relatively few cell types. Roles for GnRH receptor signaling at different stages of animal development and its influence on reproductive health remain largely unexplored, except in cases of hereditary hypogonadal infertility. In addition to regulating hormone secretion, GnRH is postulated to act as a chemokine or a growth- and differentiation-inducing factor. Hence, receptor activation may influence the function of neuronal networks in the brain and the maturation of reproductive tissue epithelia. GnRH may also potentially influence the biology of cancerous cells in reproductive tissue since receptor activation may signal terminal differentiation, cell cycle arrest or apoptosis. In this context, the cell surface expression of GnRH receptor is important since it influences the intensity of intracellular signaling, and correlates with the ability to inhibit proliferation in transformed cells in vitro. Here, we review data on the effects of GnRH agonists on cell proliferation and apoptosis, and put forward hypotheses for investigation to determine whether the GnRH receptor acts as a tumor suppressor in neuroendocrine or epithelial cells.

Identification of a novel ligand binding residue Arg38(1.35) in the human gonadotropin-releasing hormone receptor.

Delineation of peptide ligand binding sites is of fundamental importance in rational drug design and in understanding ligand-induced receptor activation. Molecular modeling and ligand docking to previously experimentally identified binding sites revealed a putative novel interaction between the C terminus of gonadotropin-releasing hormone (GnRH) and Arg(38(1.35)), located at the extracellular end of transmembrane domain 1 of the human GnRH receptor. Mutation of Arg(38(1.35)) to alanine resulted in 989- and 1268-fold reduction in affinity for GnRH I and GnRH II, respectively, the two endogenous ligands. Conservative mutation of Arg(38(1.35)) to lysine had less effect, giving reduced affinities of GnRH I and GnRH II by 24- and 54-fold, respectively. To test whether Arg(38(1.35)) interacts with the C-terminal Gly(10)-NH(2) of GnRH, binding of GnRH analogs with substitution of the C-terminal glycinamide with ethylamide ([Pro(9)-NHEt]GnRH) was studied with wild-type and Arg(38(1.35)) mutant receptors. Mutation of Arg(38(1.35)) to lysine or alanine had much smaller effect on receptor affinity for [Pro(9)-NHEt]GnRH analogs and no effect on binding affinity of peptide antagonist cetrorelix. In parallel with the decreased affinity, the mutants also gave a decreased potency to GnRH-elicited inositol phosphate (IP) responses. The mutant receptors had effects on [Pro(9)-NHEt]GnRH-elicited IP responses similar to that of the parent GnRHs. These findings indicate that Arg(38(1.35)) of the GnRH receptor is essential for high-affinity binding of GnRH agonists and stabilizing the receptor active conformation. The mutagenesis results support the prediction of molecular modeling that Arg(38(1.35)) interacts with the C-terminal glycinamide and probably forms hydrogen bonds with the backbone carbonyl of Pro(9) and Gly(10)-NH(2).

Diversity of actions of GnRHs mediated by ligand-induced selective signaling.

Geoffrey Wingfield Harris' demonstration of hypothalamic hormones regulating pituitary function led to their structural identification and therapeutic utilization in a wide spectrum of diseases. Amongst these, Gonadotropin Releasing Hormone (GnRH) and its analogs are widely employed in modulating gonadotropin and sex steroid secretion to treat infertility, precocious puberty and many hormone-dependent diseases including endometriosis, uterine fibroids and prostatic cancer. While these effects are all mediated via modulation of the pituitary gonadotrope GnRH receptor and the G(q) signaling pathway, it has become increasingly apparent that GnRH regulates many extrapituitary cells in the nervous system and periphery. This review focuses on two such examples, namely GnRH analog effects on reproductive behaviors and GnRH analog effects on the inhibition of cancer cell growth. For both effects the relative activities of a range of GnRH analogs is distinctly different from their effects on the pituitary gonadotrope and different signaling pathways are utilized. As there is only a single functional GnRH receptor type in man we have proposed that the GnRH receptor can assume different conformations which have different selectivity for GnRH analogs and intracellular signaling proteins complexes. This ligand-induced selective-signaling recruits certain pathways while by-passing others and has implications in developing more selective GnRH analogs for highly specific therapeutic intervention.

Mammalian type I gonadotropin-releasing hormone receptors undergo slow, constitutive, agonist-independent internalization.

Regulatory elements present in the cytoplasmic carboxyl-terminal tails of G protein-coupled receptors contribute to agonist-dependent receptor desensitization, internalization, and association with accessory proteins such as beta-arrestin. The mammalian type I GnRH receptors are unique among the rhodopsin-like G protein-coupled receptors because they lack a cytoplasmic carboxyl-terminal tail. In addition, they do not recruit beta-arrestin, nor do they undergo rapid desensitization. By measuring the internalization of labeled GnRH agonists, previous studies have reported that mammalian type I GnRH receptors undergo slow agonist-dependent internalization. In the present study, we have measured the internalization of epitope-tagged GnRH receptors, both in the absence and presence of GnRH stimulation. We demonstrate that mammalian type I GnRH receptors exhibit a low level of constitutive agonist-independent internalization. Stimulation with GnRH agonist did not significantly enhance the level of receptor internalization above the constitutive level. In contrast, the catfish GnRH and rat TRH receptors, which have cytoplasmic carboxyl-terminal tails, displayed similar levels of constitutive agonist-independent internalization but underwent robust agonist-dependent internalization, as did chimeras of the mammalian type I GnRH receptor with the cytoplasmic carboxyl-terminal tails of the catfish GnRH receptor or the rat TRH receptor. When the carboxyl-terminal Tyr325 and Leu328 residues of the mammalian type I GnRH receptor were replaced with alanines, these two mutant receptors underwent significantly impaired internalization, suggesting a function for the Tyr-X-X-Leu sequence in mediating the constitutive agonist-independent internalization of mammalian type I GnRH receptors. These findings provide further support for the underlying notion that the absence of the cytoplasmic carboxyl-terminal tail of the mammalian type I GnRH receptors has been selected for during evolution to prevent rapid receptor desensitization and internalization to allow protracted GnRH signaling in mammals.

Structural determinants for ligand-receptor conformational selection in a peptide G protein-coupled receptor.

G protein coupled receptors (GPCRs) modulate the majority of physiological processes through specific intermolecular interactions with structurally diverse ligands and activation of differential intracellular signaling. A key issue yet to be resolved is how GPCRs developed selectivity and diversity of ligand binding and intracellular signaling during evolution. We have explored the structural basis of selectivity of naturally occurring gonadotropin-releasing hormones (GnRHs) from different species in the single functional human GnRH receptor. We found that the highly variable amino acids in position 8 of the naturally occurring isoforms of GnRH play a discriminating role in selecting receptor conformational states. The human GnRH receptor has a higher affinity for the cognate GnRH I but a lower affinity for GnRH II and GnRHs from other species possessing substitutions for Arg(8). The latter were partial agonists in the human GnRH receptor. Mutation of Asn(7.45) in transmembrane domain (TM) 7 had no effect on GnRH I affinity but specifically increased affinity for other GnRHs and converted them to full agonists. Using molecular modeling and site-directed mutagenesis, we demonstrated that the highly conserved Asn(7.45) makes intramolecular interactions with a highly conserved Cys(6.47) in TM 6, suggesting that disruption of this intramolecular interaction induces a receptor conformational change which allosterically alters ligand specific binding sites and changes ligand selectivity and signaling efficacy. These results reveal GnRH ligand and receptor structural elements for conformational selection, and support co-evolution of GnRH ligand and receptor conformations.

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor.

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.