Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane.

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 microCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Gonadotropin-releasing hormone receptor microaggregation. Rate monitored by fluorescence resonance energy transfer.

Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release and is a therapeutic target for human and animal reproductive diseases. In the present study we have utilized the technique of fluorescence resonance energy transfer to monitor the rate of GnRH receptor-receptor interactions. This technique relies on the observation that the degree of physical intimacy of molecules can be assessed by the tendency of proximal fluorophores to exchange energy. Our data indicate that GnRH agonist, but not antagonist, occupancy of the GnRH receptor promotes physical intimacy (microaggregation) between receptors. The time course indicates that this occurs promptly (<1 min) after occupancy and persists for at least 80 min and within the physiologically relevant range of the releasing hormone. The process measured is not inhibited by 0.1 mm vinblastin, 2 microm cytochalasin D, or 3 mm EGTA, an observation that distinguishes it from macroaggregation (patching, capping, and internalization). These observations, along with reports from other laboratories, are consonant with a growing body of evidence that indicates that microaggregation is an early event following agonist occupancy of the receptor and part of the mechanism by which effector regulation occurs.

Molecular cloning and expression of a type-two somatostatin receptor in goldfish brain and pituitary.

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, a SRIF receptor cDNA was cloned and sequenced from goldfish brain using PCR and cDNA library screening. The cDNA encodes a 380-amino acid goldfish type-two SRIF receptor (designated as sst(2)), with seven putative transmembrane domains (TMD) and YANSCANP motif in the seventh TMD, a signature sequence for the mammalian SRIF receptor (sst) family. In addition, the amino acid sequence of the receptor has 61-62% homology to mammalian sst(2), 41-47% homology to other mammalian sst subtypes and 41-43% homology to recently identified fish sst(1) and sst(3) receptors. Both SRIF-14 and [Pro(2)]SRIF-14, two of the native goldfish SRIF forms, but not a putative goldfish SRIF-28, significantly inhibited forskolin-stimulated adenosine 3':5'-cyclic monophosphate (cAMP) release in COS-7 cells transiently expressing goldfish sst(2), suggesting functional coupling of the receptor to adenylate cyclase. None of the three peptides affected inositol phosphate production in the same receptor expression system. Northern blot showed that mRNA for the sst(2) receptor is widely distributed in goldfish brain, and highly expressed in the pituitary. The decrease in pituitary sst(2) mRNA levels following estradiol implantation suggests the presence of a negative feedback mechanism on sst(2) gene expression.

A mathematical model quantifying GnRH-induced LH secretion from gonadotropes.

A mathematical model is developed to investigate the rate of release of luteinizing hormone (LH) from pituitary gonadotropes in response to short pulses of gonadotropin-releasing hormone (GnRH). The model includes binding of the hormone to its receptor, dimerization, interaction with a G protein, production of inositol 1,4, 5-trisphosphate, release of Ca(2+) from the endoplasmic reticulum, entrance of Ca(2+) into the cytosol via voltage-gated membrane channels, pumping of Ca(2+) out of the cytosol via membrane and endoplasmic reticulum pumps, and release of LH. Cytosolic Ca(2+) dynamics are simplified (i.e., oscillations are not included in the model), and it is assumed that there is only one pool of releasable LH. Despite these and other simplifications, the model explains the qualitative features of LH release in response to GnRH pulses of various durations and different concentrations in the presence and absence of external Ca(2+).

Combined modification of intracellular and extracellular loci on human gonadotropin-releasing hormone receptor provides a mechanism for enhanced expression.

The mammalian gonadotropin-releasing hormone (GnRH) receptor (GnRH-R) has been a therapeutic target for human and animal medicine. This receptor is a unique G-protein-coupled receptor that lacks the intracellular C-terminal domain commonly associated with this family. Development of highthrough put screens for agents active in humans has been hampered by low expression levels of the hGnRH-R in cellular models. Two sites have attracted the interest of laboratories studying regulation of expression. The chimeric addition of the C-terminal tail from catfish GnRH-R (cfGnRH-R) to the rat GnRH-R significantly augmented receptor expression in GH3 cells. In addition, rodent GnRH-R contains 327 amino acids, but cow, sheep, and human GnRH-R (hGnRH-R) contain 328 residues, the "additional" residue being a Lys 191. Deletion of Lys 191 (del 191) from the hGnRH-R resulted in increased receptor expression levels and decreased internalization rates in both COS-7 and HEK 293 cells. In this study, the combined effect of the addition of the C-tail from cfGnRH-R and deletion of the Lys 191 from the hGnRH-R was compared to expression of the wild-type (WT) or either alteration alone in a transient expression system using primate cells. The altered receptor (hGnRH-R[del 191]-C-tail) showed significantly increased receptor expression at the cell surface compared with the WT or either modification alone. The inositol phosphate response to stimulation was also significantly elevated in response to GnRH agonist. After treatment with a GnRH agonist, the altered receptors showed a slower internalization rate. The homologous steady-state regulation of the WT and the altered receptors was similar, although the response of the altered receptors was significantly decreased. These results suggest that the conformational change in the receptor as a result of the deletion of Lys 191 and the addition of the C-terminus tail substantially increased the steady-state receptor expression and decreased internalization and homologous regulation. Because the effects on expression are greater than additive, it appears that these alterations exert their effects by differing means. These techniques for expression of the hGnRH-R in transfected mammalian cells provide the basis for a therapeutic screen for GnRH analogs, agonists, and antagonists of the hGnRH.

Molecular cloning and expression of two type one somatostatin receptors in goldfish brain.

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, two SRIF receptor complementary DNAs (cDNAs) were cloned and sequenced from goldfish brain using PCR and cDNA library screening. The two cDNAs share 92% similarity in nucleotide sequence and 98% similarity in the deduced amino acid sequences and are presumably derived from duplicate genes, as goldfish are tetraploid. Two cDNAs encode two 367-amino acid goldfish type one SRIF receptors (designated as sst1A and sst1B, respectively), with seven putative transmembrane domains (TMD) and YANSCANP motif in the 7th TMD, a signature sequence for mammalian SRIF receptor (sst) family. In addition, the amino acid sequences of two receptors have 76% and 75% similarity to human or rat sst1, respectively, and 39-55% similarities to other mammalian sst subtypes (sst2-5), suggesting that the two receptors could be the goldfish homologs of mammalian sst1. The difference between goldfish and mammalian sst1 is mainly reflected by the extreme divergence in their extracellular N termini. Both SRIF-14 and [Pro2]SRIF-14, two of the native goldfish SRIF forms, significantly inhibited forskolin-stimulated cAMP release in COS-7 cells transiently expressing goldfish sSt1A or sst1B, suggesting functional coupling of the two receptors to adenylate cyclase. Northern blot and RT-PCR showed that messenger RNAs (mRNAs) for both receptors are widely distributed throughout goldfish brain, whereas only one receptor mRNA is expressed in the pituitary. RT-PCR analysis also detected sst1 receptor mRNAs in several peripheral tissues. These findings provide fundamental information for studying the mechanism of SRIF actions in vertebrates and structural analysis of mammalian sst receptors.

Simultaneous and independent visualization of the gonadotropin-releasing hormone receptor and its ligand: evidence for independent processing and recycling in living cells.

The first step in GnRH signaling is binding by the peptide to its plasma membrane receptor (GnRHR). The receptor is a member of the seven transmembrane G protein-coupled class but lacks the characteristic C-terminal cytoplasmic tail, making it among the smallest receptors in this superfamily. It has been known since 1980 that agonist occupancy of the GnRHR results in patching, capping, and internalization, although it has not been possible to localize the unoccupied GnRHR, because elaboration of receptor antisera has not been easy to achieve. The recent production of a green fluorescent protein (GFP) conjugate of the GnRHR ("rGnRHR-C-tail-GFP") that is expressed in cells, targeted to the plasma membrane, binds GnRH analogs and couples to G proteins has made it possible to monitor movement of the unoccupied receptor by confocal microscopy. In the present study, we used this probe, along with Texas Red conjugates of a GnRH agonist, to examine simultaneous processing of the receptor and its ligands. The preparation of the GFP GnRHR chimera has been described. A Texas Red conjugate was made from the GnRH agonist D-Lys6-Pro9-des-Gly10EA-GnRH by standard procedures. Bioactivity of this conjugate was confirmed. Confocal fluorescence images of living GGH3 cells showed that the agonist binds the GFP-GnRH receptor construct on the cell membrane and causes the internalization of vesicles delimited by a membrane. Shortly after internalization, the agonist separates from receptor inside the vesicle, although it is still enclosed in membranes containing free receptor. As the vesicles approach the perinuclear space, the separation between receptor and agonist is more pronounced. Free receptor appears at the cell membrane after the internalization of agonist has been completed. The protein synthesis inhibitor, cycloheximide (1 mM) did not inhibit this process, suggesting that the free receptor results from the recycling of previously internalized vesicles rather than from newly synthesized receptor. These studies show visual evidence for recycling of the GnRH receptor in cultured cells.

Gonadotropin-releasing hormone receptor concentration differentially regulates intracellular signaling pathways in GGH3 cells.

Pituitary cell lines (GGH3) expressing the GnRH receptor (GnRHR) were used to investigate the effect of GnRHR concentration on the ability of a GnRH agonist to activate second messenger systems. Four different strategies were utilized to generate cells expressing functionally different concentrations of receptors: (1) transient transfection with different concentrations of wild type GnRHR into GH3 cells, (2) utilization of two cell lines derived from a common stably transfected line expressing high (4,209 +/- 535 receptors/cell) or low (1,031 +/- 36 receptors/cell) concentrations of GnRHR, (3) co-incubation of GGH3-1' cells with a GnRH agonist (Buserelin) and a GnRH antagonist to compete for binding sites, and (4) photo-affinity binding to GnRHR with a GnRH antagonist to change effective receptor concentration. A range of receptor concentrations (1,000-8,000 receptors/cell) were generated by these techniques. Inositol phosphate (IP) and cAMP accumulation were quantified to assess the effect of receptor concentration on receptor-effector coupling. Under all four paradigms, the efficacy and potency of Buserelin stimulated IP production was dependent on receptor concentration. In contrast, Buserelin stimulated cAMP release was relatively unchanged at varying concentrations of GnRHR. This suggests that the cellular concentration of GnRHR affects the induction of cell signaling pathways. These results demonstrate that a single ligand-receptor-complex can differentially activate second messenger systems and present a mechanism by which multiple physiological endpoints can be differentially regulated by a single hormone/receptor interaction.

Structure-activity relationships of G protein-coupled receptors.

The primary function of cell-surface receptors is to discriminate the specific signaling molecule or ligand from a large array of chemically diverse extracellular substances and to activate an effector signaling cascade that triggers an intracellular response and eventually a biological effect. G protein-coupled cell-surface receptors (GPCRs) mediate their intracellular actions through the activation of guanine nucleotide-binding signal-transducing proteins (G proteins), which form a diverse family of regulatory GTPases that, in the GTP-bound state, bind and activate downstream membrane-localized effectors. Hundreds of GPCRs signal through one or more of these G proteins in response to a large variety of stimuli including photons, neurotransmitters, and hormones of variable molecular structure. The mechanisms by which these ligands provoke activation of the receptor/G-protein system are highly complex and multifactorial. Knowledge and mapping of the structural determinants and requirements for optimal GPCR function are of paramount importance, not only for a better and more detailed understanding of the molecular basis of ligand action and receptor function in normal and abnormal conditions, but also for a rational design of early diagnostic and therapeutic tools that may allow exogenous regulation of receptor and G protein function in disease processes.

Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone.

A central question in endocrinology is how a single ligand interacting with a single receptor can mediate multiple responses. GnRH interaction with receptor offers a prime example, leading to the regulation of synthesis and release of at least three molecules, regulation of target cell responsiveness and receptor number. The present study suggests a molecular model consistent with extant data that provides a mechanism by which this may occur and, further, which allows for coordinate regulation.

Mutations at the consensus phosphorylation sites in the third intracellular loop of the rat gonadotropin-releasing hormone receptor: effects on receptor ligand binding and signal transduction.

In this study, site-directed mutagenesis of potential phosphorylation sites (Thr238, Ser253, and Thr264) for protein kinase C and C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rat GnRH receptor (rGnRHR) was performed to assess the significance of these regions in the function of the GnRHR. Mutation at one or all of the three potential phosphorylation sites had differential effects on receptor ligand binding. Mutation of Ser253 or Thr264 to Ala did not significantly affect the receptor-binding affinity but decreased the number of measurable binding sites. Mutation of Thr238 to Ala or triple mutation of Thr238, Ser253, and Thr264 impaired or abolished receptor-binding affinity. Mutations of the potential phosphorylation sites affected receptor-mediated inositol phospholipid (IP) production and correlated with alterations in receptor binding after mutation, but they did not significantly affect receptor-mediated cAMP production or cAMP-mediated prolactin release. In addition, mutation of Ser253 or Thr264 to Ala did not affect the GnRH-provoked desensitization in terms of GnRH agonist-stimulated IP production. Deletion of the C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rGnRHR, including a potential phosphorylation site (Thr264), abolished the receptor-binding affinity and receptor-mediated signal transduction. Replacement of the deleted C-terminal portion with a C-terminal portion (Ala-Ala-Arg-Thr-Leu-Ser) of the third intracellular loop of the Gq/11-coupled rat M1 muscarinic acetylcholine receptor did not restore receptor function. These results suggest that the potential phosphorylation sites or the region around the phosphorylation site of the third intracellular loop of the GnRHR is important for the structural integrity and expression of the receptor but that phosphorylation at these sites is not required for desensitization.

Gonadotropin and gonadal steroid release in response to a gonadotropin-releasing hormone agonist in Gqalpha and G11alpha knockout mice.

In this study, we used mice lacking the G11alpha [G11 knockout (KO)] or Gqalpha gene (Gq KO) to examine LH release in response to a metabolically stable GnRH agonist (Buserelin). Mice homozygous for the absence of G11alpha and Gqalpha appear to breed normally. Treatment of (5 wk old) female KO mice with the GnRH agonist Buserelin (2 microg/100 microl, sc) resulted in a rapid increase of serum LH levels (reaching 328 +/- 58 pg/25 microl for G11 KO; 739 +/- 95 pg/25 microl for Gq KO) at 75 min. Similar treatment of the control strain, 129SvEvTacfBr for G11 KO or the heterozygous mice for Gq KO, resulted in an increase in serum LH levels (428 +/- 57 pg/25 microl for G11 KO; 884 +/- 31 pg/25 microl for Gq KO) at 75 min. Both G11 KO and Gq KO male mice released LH in response to Buserelin (2 microg/100 microl of vehicle; 363 +/- 53 pg/25 microl and 749 +/- 50 pg/25 microl 1 h after treatment, respectively). These values were not significantly different from the control strain. In a long-term experiment, Buserelin was administered every 12 h, and LH release was assayed 1 h later. In female G11 KO mice and control strain, serum LH levels reached approximately 500 pg/25 microl within the first hour, then subsided to a steady level (approximately 100 pg/25 microl) for 109 h. In male G11 KO mice and in control strain, elevated LH release lasted for 13 h; however, LH levels in the G11 KO male mice did not reach control levels for approximately 49 h. In a similar experimental protocol, the Gq KO male mice released less LH (531 +/- 95 pg/25 microl) after 13 h from the start of treatment than the heterozygous male mice (865 +/- 57 pg/25 microl), but the female KO mice released more LH (634 +/- 56 pg/25 microl) after 1 h from the start of treatment than the heterozygous female mice (346 +/- 63 pg/25 microl). However, after the initial LH flare, the LH levels in the heterozygous mice never reached the basal levels achieved by the KO mice. G11 KO mice were less sensitive to low doses (5 ng/per animal) of Buserelin than the respective control mice. Male G11 KO mice produced more testosterone than the control mice after 1 h of stimulation by 2 microg of Buserelin, whereas there was no significant difference in Buserelin stimulated testosterone levels between Gq KO and heterozygous control mice. There was no significant difference in Buserelin stimulated estradiol production in the female Gq KO mice compared with control groups of mice. However, female G11 KO mice produced less estradiol in response to Buserelin (2 microg) compared with control strain. Although there were differences in the dynamics of LH release and steroid production in response to Buserelin treatment compared with control groups of mice, the lack of complete abolition of these processes, such as stimulated LH release, and steroid production, suggests that these G proteins are either not absolutely required or are able to functionally compensate for each other.

The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to Gs- and G(q/11)-mediated signal transduction pathways: evidence from loop fragment transfection in GGH3 cells.

The GnRH receptor (GnRH-R) belongs to the rhodopsin/beta-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane-proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH(3)1' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both Gs and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other Gs-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH(3)1' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH(3)1' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the alpha1B-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach-muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the Gs protein). Transfection of loop 3i from the D1A -dopamine receptor (coupled to the Gs protein) produced a selective attenuation (40%) in Gs-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and alpha2A-adrenergic receptors). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH(3)1' cell line, this loop is also involved in signal transduction mediated through the Gs protein pathway.

Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation.

Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3- to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGnRHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of down-regulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10[-13]-10[-9] M). GH3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10[-13]-10[-7] M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins.

Redistribution of G(q/11)alpha in the pituitary gonadotrope in response to a gonadotropin-releasing hormone agonist.

In the present study, we took advantage of high-resolution multilaser confocal microscopy to examine the distribution of the alpha-subunit of the guanyl nucleotide binding protein subfamily G(q/11) (G(q/11)alpha). Dispersed cultures of pituitary cells were prepared from female weanling rats, fixed, permeabilized, and then stained with monoclonal antiserum (mouse) to the gonadotrope-specific form of secretogranin (SIIp), which was then tagged with Texas Red. Accordingly, the subpopulation of gonadotropes (approximately 15% of total cells) could be identified against a background of other pituitary cell types. G(q/11)alpha was localized with antiserum made in rabbit, then tagged with fluorescein. Hoechst 33258 nuclear stain was also used in some experiments for topological reference. The data indicate localization of the G(q/11)alpha in a cellular region near the plasma membrane and external to the border of the layer occupied by secretory granules. In the absence of activation, there were an average of six clusters of G(q/11)alpha in a section 1 microm thick and through the center of the cell. This corresponds to an average of 60 clusters per cell, assuming a mean gonadotrope diameter of 10 microm. Following continuous treatment with 0.1 microg/ml Buserelin, a metabolically stable GnRH agonist, the average number of clusters increased to 200/cell after 40 min and remained approximately constant for 120 min. This increase was blocked by the protein synthesis inhibitor, cycloheximide. In response to Buserelin, there was an additional increase in the number of clusters inside the cell in the area occupied by the secretory granules and in the perinuclear area. Prolonged (24 h) treatment with Buserelin, sufficient to provoke the onset of desensitization, did not significantly change total numbers of G(q/11)alpha clusters, although more were located in the peripheral compartment, an increase that occurred at the expense of the cytoplasmic compartment. Redistribution of the G(q/11)alpha family may be functionally significant, because this moiety may be rate limiting at the site of regulation of signal transduction.

Visualization of unoccupied and occupied gonadotropin-releasing hormone receptors in living cells.

Three chimeras of the rat GnRH receptor (rGnRHR) and an enhanced green fluorescent protein (GFP) were assessed to examine their suitability as probes of the receptor in transfected GH3 cells. Direct fusion of GFP to the N or C terminus of the rGnRHR abolished the receptor ligand binding affinity and the chimeric receptors were intracellularly localized. In contrast, rGnRHR-Ctail-GFP, a fusion of the N-terminus of the GFP to the C-terminus of the rGnRHR with the intracellular C-terminal tail of the catfish GnRHR as an intermediate spacer, was functional in terms of plasma membrane localization, ligand binding ability, receptor-mediated signal transduction and pattern of homologous down-regulation. The functional chimera of GnRHR and GFP provided a useful model for observation of GnRHR distribution and agonist-stimulated trafficking in living cells.

Regulation of G(q/11)alpha by the gonadotropin-releasing hormone receptor.

Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH-stimulated palmitoylation of G(q/11)alpha to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-(3)H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G(q/11)alpha. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0-120 min) labeling of G(q/11)alpha. GnRH (10(-12), 10(-10), 10(-8), or 10(-6) g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)alpha compared with controls. Cholera toxin (5 microg/ml; activator of G(i)alpha), pertussis toxin (100 ng/ml; inhibitor of G(i)alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G(q/11)alpha above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G(q/11)alpha above basal levels, but not to the same extent as 10(-6) g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of alpha1B-adrenergic receptor (alpha1B 3i) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha1B 3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)alpha), resulted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of alpha2A adrenergic receptor, M2-acetylcholine receptor (both couple to G(i)alpha), and D1A-receptor (couples to G(s)alpha) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of G(q/11)alpha in a PRL-secreting adenoma cell line (GGH(3)1') expressing the GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 microg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15-20% decrease in total G(q/11)alpha at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10(-6) g/ml). Neither Antide (10(-6) g/ml, 24 h) alone nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The loss of G(q/11)alpha immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH3 cells.

Brain gonadotropin releasing hormone receptors: localization and regulation.

In this review, the current information about the location of GnRH receptor protein and GnRH receptor mRNA in the rat central nervous system is summarized as well as the changes that occur in the GnRH receptor mRNA levels during different endocrine conditions of the animals. The results of these studies show that GnRH receptor protein and mRNA levels change in parallel in the hippocampus, suggesting that pretranscriptional factors control the synthesis of the receptor. In the arcuate and ventromedial nuclei of the hypothalamus, GnRH receptor mRNA levels are highest during the early morning of proestrus and during the morning of an estrogen-progesterone-induced LH surge. The timing of the changes in GnRH receptor mRNA levels indicates that increasing levels of estradiol are responsible for the increase in GnRH receptor synthesis. Binding of GnRH agonist to the brain GnRH receptor causes a dose-dependent increase in inositol phosphates as well as changes in intracellular Ca++ levels of the target neurons. Together, it is suggested that GnRH functions in the brain as a neurotransmitter and/or modulator linking the peripheral endocrine effects of GnRH to actions of the peptide inside the central nervous system where it can facilitate, for example, reproductive behaviors.

Gonadotropin releasing hormone agonist provokes homologous receptor microaggregation: an early event in seven-transmembrane receptor mediated signaling.

Gonadotropin releasing hormone (GnRH) mediates interactions between the neural and the endocrine systems. The GnRH receptor is a member of the ubiquitous seven-transmembrane segment G-protein-coupled receptor class and is the target of drug development for treatment of breast and prostate cancer, regulation of fertility, endometriosis and a range of other medical and veterinary uses. This study shows that occupancy of the receptor by an agonist (but not an antagonist) promotes receptor-receptor interactions which appears to be an early event in hormone action.