Gonadotropin-releasing hormone receptor expression in Xenopus oocytes

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.     

Molecular characterization of a functional cDNA for rat substance P receptor

This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor cDNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptors are very similar in both size and amino acid sequences, particularly in the putative transmembrane regions and the first and second cytoplasmic loops. This similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarity and divergence would thus contribute to the expression of similar but pharmacologically distinguishable activities of the two tachykinin receptors.     

Cloning, sequencing and tissue distribution of a candidate G protein-coupled receptor from rat pituitary gland.

A new member of the family of G protein-coupled receptors has been isolated from a rat pituitary cDNA library by the polymerase chain reaction (PCR) using degenerate oligonucleotide primers. The corresponding protein sequence shows seven transmembrane domains and contains conserved regions of homology characteristic of the G protein-coupled class of receptors. The novel receptor mRNA is expressed in the brain, pituitary gland and testis, and has been localized by in situ hybridization in discrete regions of the brain. Expression of the receptor mRNA in Xenopus oocytes and in transfected mammalian cells has not yet permitted identification of the corresponding ligand for this receptor.     

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.     

A study on the localization and distribution of GnRH and its receptor in rat submaxillary glands by immunohistochemical,in situ hybridization and RT-PCR

We investigated the rat submaxillary gland for the presence of GnRH and GnRH receptors, the localization and colocalization of GnRH, GnRH receptor and their mRNA, and studied the sequence of GnRH receptor complementary DNA (cDNA) by immunohistochemistry, in situ hybridization and RT-PCR. The results showed that GnRH and GnRH receptor immunoreactive materials were colocalized in the epithelial cells of the serous acinus and glandular duct. The GnRH and GnRH receptor mRNA hybridization signals were detected in the above cells. The sequence obtained from the RT-PCR product was identical to the published cDNA sequence of GnRH receptor in the rat pituitary. The results suggested that the rat submaxillary gland was capable of synthesizing GnRH and GnRH receptors. GnRH may be involved in the functional regulation of the submaxillary gland through autocrine or paracrine activity.     

Constitutive signaling by Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor desensitizes calcium mobilization by other receptors

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux. In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools.     

Mammalian gonadotropin-releasing hormone (GnRH) receptor subtypes

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that governs gonadotropin secretion through interaction with its seven transmembrane (7TM), G protein-coupled receptor (GPCR) expressed by anterior pituitary cells. A second decapeptide, GnRH II, originally discovered in the chicken hypothalamus was recently reported to be expressed in the mammalian hypothalamus as well. A search of the recently-sequenced human genome identified a 7TM/GPCR on chromosome 1 that exhibited a higher identity with non-mammalian vertebrate GnRH II receptors (55%) than with the human GnRH I receptor (39%). Molecular cloning and nucleotide sequencing of this putative GnRH II receptor cDNA from monkey pituitary gland revealed a 379 amino acid receptor that, unlike the GnRH I receptor, possessed a C-terminal tail. Heterologous expression and functional testing of the receptor in COS-1 cells confirmed its identity as a GnRH II receptor: measurement of 3H-inositol phosphate accumulation revealed EC(50)s for GnRH II of 0.86 nM and for GnRH I of 337 nM. Ubiquitous tissue expression of GnRH II receptor mRNA was observed using a human tissue RNA expression array and a 32P-labeled antisense riboprobe representing the 7TM region of human GnRH II receptor cDNA. As predicted by the presence of its C-terminal tail, the GnRH II receptor was desensitized by GnRH II treatment whereas the naturally tail-less GnRH I receptor was not desensitized by GnRH I. Pharmacological analysis of the GnRH II receptor revealed that GnRH I 'superagonists' were more potent than GnRH I but less potent than GnRH II. Numerous GnRH I antagonists showed neither antagonistic nor agonistic activity with the GnRH II receptor. The functions of the GnRH II receptor are unknown but may include regulation of gonadotropin secretion, female sexual behavior, or tumor cell growth.     

Structure and functional expression of a cloned Xenopus thyroid hormone receptor.

A clone corresponding to a thyroid hormone receptor was isolated from a Xenopus laevis cDNA library prepared from folliculated oocytes. The cDNA encodes a protein of 418 amino acid residues with a domain structure, including a putative DNA binding region with two zinc fingers, similar to other members of the v-erbA-related superfamily of receptors. The encoded protein resembles the TR alpha 1-type receptor of the rat. When expressed in COS cells the protein product binds triiodothyronine with a Kd of 0.12 nM. The receptor mediates thyroid-hormone-inducible expression of a reporter gene which includes a thyroid hormone response element in its upstream region.     

Simple luminometric assay to detect phosphoinositol-linked receptor expression in Xenopus oocytes.

A simple and sensitive method to measure the expression of phosphoinositol-linked receptors in Xenopus laevis oocytes is described. Oocytes are co-injected with the calcium photoprotein aequorin and RNA, encoding the receptor of interest. The binding of ligand to the expressed receptor increases intracellular calcium that induces the aequorin to luminesce. With an autosampler-equipped luminometer, this provides a fully automated assay of receptor expression of oocytes. This method was applied to cloning the bombesin/GRP receptor expressed in Swiss 3T3 fibroblasts. Oocytes expressing the cloned BR showed up to a 10,000-fold increase in light emission in response to bombesin. The sensitivity of this procedure allowed detection of positive luminometer signals in single oocytes injected with RNA transcribed from cDNA pools as large as 25,000 clones. These findings show the potential value of this procedure for rapid screening of expression libraries, structure/function analysis of receptors and analysis of receptor antagonists or agonists.     

The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit

Sodium channels consist of a pore-forming alpha subunit and auxiliary beta 1 and beta 2 subunits. The subunit beta 1 alters the kinetics and voltage-dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N-terminal extracellular domain of beta 1, but does not require the intracellular C-terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain Na(V)1.2a sodium channel alpha subunits expressed in Chinese hamster lung cells by a mutant beta1 construct with 34 amino acids deleted from the C-terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co-immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant beta 1 with alpha, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10,000 times more mutant beta 1 RNA was required to achieve the level of functional modulation observed with injection of full-length beta 1. Together, these findings suggest that the C-terminal cytoplasmic domain of beta 1 is an important determinant of beta1 binding to the sodium channel alpha subunit in both mammalian cells and Xenopus oocytes.     

Structure and functional expression of the cloned rat neurotensin receptor.

A functional cDNA clone for the rat neurotensin receptor was isolated by combining molecular cloning in an RNA expression vector with an electrophysiological assay in Xenopus oocytes. The neurotensin receptor consists of 424 amino acids with seven putative transmembrane domains and belongs to the family of G protein-coupled receptors. The cloned receptor expressed in mammalian cells or in Xenopus oocytes shows a selective and high-affinity binding to neurotensin peptides and undergoes potent desensitization by repeated application of neurotensin. The neurotensin receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. This investigation discloses the molecular nature of the neurotensin receptor, which mediates the diverse neuronal and peripheral actions of neurotensin by effecting the G protein-associated second messenger system.     

Intracellular responses to gonadotropin-releasing hormone in a clonal cell line of the gonadotrope lineage.

We recently derived a GnRH-responsive pituitary cell line of the gonadotrope lineage (alpha T3-1) by targeted oncogenesis in transgenic mice. Here, we report studies characterizing the GnRH receptors present in these cells and the intracellular responses to GnRH treatment. The receptors in alpha T3-1 cells show specificity for different GnRH analogs, with dissociation constants very similar to those found in normal rat and mouse pituitary. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists increases phosphoinositide turnover and protein kinase-C translocation to membranes, and enhances activation of voltage-sensitive calcium channels. However, GnRH does not affect cAMP levels. Analysis of alpha-subunit mRNA levels demonstrated induction by GnRH and phorbol esters. Our results indicate that GnRH initiates a cascade of intracellular events that generate a set of second messengers, one or more of which is involved in the regulation of gene expression. The responses of alpha T3-1 cells to GnRH appear to have characteristics equivalent to those of primary pituitary gonadotropes, indicating the utility of this cell line as a model system for the study of GnRH responses.     

Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor.

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling.     

Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus.

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.     

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1)

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.     

Expression of functional thrombin receptors in xenopus oocytes injected with human endothelial cell mRNA.

Human endothelial cell thrombin receptors were functionally expressed in Xenopus laevis oocytes by injection of RNA extracted from human umbilical vein endothelial cells. Oocytes injected with endothelial cell RNA responded to thrombin with a Ca2(+)-dependent depolarizing current whose size depended on the amount of RNA injected. In oocytes expressing thrombin receptors, thrombin caused homologous but not heterologous desensitization. Both the catalytic and anion-binding exosites of thrombin were necessary to elicit depolarizing currents. Thus, Xenopus laevis oocytes injected with mRNA from human endothelial cells express Ca2(+)-dependent thrombin receptors which share many common features with thrombin receptors on intact endothelial cells. Xenopus oocytes may, therefore, be used as a screening system in the expression cloning of the endothelial cell thrombin receptor.     

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the β-amino acid agonist, taurine

The beta-amino acid, taurine, is a full agonist of the human glycine receptor alpha1 subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta-amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta-amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta-amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.