Molecular characterization of dopamine D2 receptor isoforms tagged with green fluorescent protein

In this study, a cleavable signal peptide fused to the enhanced green fluorescent protein (EGFP) was tagged to the extracellular N-terminus of the human dopamine D2 receptor short and long isoforms (D2S and D2L). Ligand-binding properties of EGFP-tagged receptors were essentially unchanged in comparison to their respective wild-type receptors. The dopamine-mediated activation of both EGFP-D2 isoforms generated a robust inhibition of adenylyl cyclase type 5 in intact cells. In addition, the receptor density of EGFP-D2S and EGFP-D2L in transfected human embryonic kidney 293 (HEK293) cells was not altered when compared to cells transfected with the untagged D2S and D2L. However, the receptor densities of untagged and EGFP-tagged D2L were significantly lower in comparison to those measured with D2S constructs. Moreover, the receptor density of EGFP-D2S and EGFP-D2L was differentially upregulated in cells treated with antipsychotic drugs. As assessed by confocal microscopy, both EGFP-D2 isoforms were present on the cell surface. Notably, in contrast to the predominant plasma membrane localization of EGFP-D2S, EGFP-D2L was visualized both on the plasma membrane and intracellularly before dopamine exposure. Importantly, EGFP-D2S and EGFP-D2L are robustly internalized after dopamine treatment. Overall, our data suggest a differential intracellular sorting of D2S and D2L.     

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes

Dopamine D2_LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor–receptor interactions. In the current study the existence of D2_L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist ³H-raclopride binding sites and significantly reduced the pK_iH values of the high affinity D2R agonist binding sites in ³H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2_LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists.     

Role of phospholipase D2 in the agonist-induced and constitutive endocytosis of G-protein coupled receptors

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.     

The co-existence of two growth hormone receptors and their differential expression profiles between female and male tongue sole (Cynoglossus semilaevis)

Growth hormone receptor (Ghr) is a single-transmembrane pass protein which is important in initiating the ability of growth hormone (Gh) to regulate development and somatic growth in vertebrates. In this study, molecular cloning, expression analysis of two different ghr genes (ghr1 and ghr2) in the tongue sole (Cynoglossus semilaevis) was conducted. As a result, the ghr1 and ghr2 cDNA sequences are 2364 bp and 3125 bp, each of which encodes a transmembrane protein of 633 and 561 amino acids (aa), respectively. Besides, the ghr1 gene includes nine exons and eight introns. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. Both the ghr1 and ghr2 were predominantly expressed in the liver, and the ghr1 expression level in normal males was 1.6 and 1.4 times as much as those in females and extra-large males, while the ghr2 mRNA expression level in normal males was 1.1 and 1.2 times as much as those in females and extra-large males, respectively. Ontogenetic expression analysis at early life stages indicated that the ghr1 and ghr2 mRNAs were detected at all of the 35 sampling points (from oosphere to 410 days-old). Furthermore, the sex differences in ghr mRNA expressions were also examined by using a full-sib family of C. semilaevis. Significantly higher levels of ghr1 mRNA were observed in males than in females at most stages of the sampling period (P < 0.01). The ghr2 mRNA expression at most stages exhibited a significant sexual difference at each sampling point (P < 0.01) without any variation trend related with the sexes during the whole sampling period.     

Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes

The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226, and eLDHB lost negatively charged Glu 14, as compared to the highly conserved residues at these positions in the corresponding amino acid sequences of other mammals. These alterations were identified in six equine species by genomic DNA analysis. A comparison of the equine and human 3D structures revealed that the substituted His 88 and 226 of the eLDHA monomer and the deleted Glu 14 of the eLDHB monomer altered the surface charge of equine LDH tetramers and that these three residues were located in important regions affecting the catalytic kinetics. Also, RT-PCR amplification of the three myosin heavy chain isoforms corroborated that the cervical muscle as postural muscle of the thoroughbred horse was composed of more oxidative myofibers than the dynamic muscle. Based on this property, the mRNA expression patterns of eLDHA, eLDHB, and eGAPDH in various tissues were analyzed by using real-time PCR. The expression levels of these three genes in the cervical muscle were not always relatively higher than in the brain or heart.     

Measurement of dopamine D2-like receptors in postmortem CNS and pituitary: differential regional changes in schizophrenia

In situ radioligand binding with autoradiography and anti-human dopamine D(2) receptor antibodies with Western blots have been used to measure the density of dopamine D(2)-like receptors in the caudate-putamen and pituitary from schizophrenic subjects who did or did not have residual antipsychotic drugs in their tissue at death. There was a significant decrease in the Ki for haloperidol displaceable [(125)I]iodosulpride binding in the pituitary (p < 0.01) and caudate-putamen (p < 0.05) from subjects with schizophrenia with residual drugs in their tissue. There was a significant decrease in the density of [(125)I]iodosulpride in the pituitary (p < 0.001) and a strong trend to a decrease in binding in the caudate-putamen (p = 0.055) from subjects with schizophrenia. By contrast, [(3)H]spiperone binding was decreased in the caudate-putamen (p < 0.05) with a trend to decreased binding in the pituitary (p = 0.07) from subjects with schizophrenia. There was no difference in the density of dopamine D(2) receptors in the caudate-putamen from subjects with schizophrenia (p = 0.31). All the findings on receptor densities were independent of drug status. [(125)I]iodosulpride binds to the dopamine D(2&3) receptors. We have shown that there is no change in the dopamine D(2) receptor in the caudate-putamen from subjects with schizophrenia and therefore, these data would be consistent with there being a decrease in the dopamine D(3) in the caudate-putamen from subjects with schizophrenia. Since dopamine D(3) receptors are absent or present at low concentrations in the pituitary, our data would suggest the dopamine D(2) receptor is decreased in that tissue from schizophrenic subjects.     

Differential voltage-sensitivity of D2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D_2L receptor and we now extend our studies to include the other members of the D₂-like receptor subfamily; the D_2S, D₃, and D₄ dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D_2S, D₃, or D₄ receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D_2S and D₄ upon depolarization. In contrast, the concentration-response relationships for D₃-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.     

Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris

The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.     

Molecular cloning and mRNA expression analysis of ornithine decarboxylase antizyme 2 in ovarian follicles of the Sichuan white goose (Anser cygnoides)

The ornithine decarboxylase antizyme 2 (OAZ2) gene is a member of the antizyme gene family. Antizymes play pivotal roles in various cellular pathways, including polyamine anabolism and apoptosis. The molecular structure and expression profile of the OAZ2 in goose ovarian follicles have not been reported. In this study, the OAZ2 cDNA sequence of the Sichuan white goose was cloned (Anser cygnoides), and phylogenetic and structural analyses of the OAZ2 were performed. The expression profiling of OAZ2 mRNA in goose ovarian follicles was examined using quantitative real-time PCR. The sequence analysis showed that the 756 bp OAZ2 sequence contained two overlapping open reading frames (ORF). ORF1 was 99 bp in length, and encoded a 32 aa polypeptide. ORF2 was 477 bp in length, and encoded a 158 aa polypeptide. The frameshift site that initiates the translation of ORF2 was located at nucleotide position 97 in the OAZ2. The analysis of OAZ2 mRNA expression in hierarchical follicles showed that the level of OAZ2 mRNA was higher in the SWF and F2 follicular stages than that in the ovarian stroma (P < 0.05). The lowest level of OAZ2 expression was detected in the ovarian stroma. These results suggest that the highly conserved frameshift region plays an important role in sustaining the function of OAZs. Furthermore, the significantly higher level of OAZ2 mRNA in the SWF stage indicates that OAZ2 may be involved in recruiting hierarchical follicles. Our results also suggest that OAZ2 may augment the effects of OAZ1 in follicle development.     

Induction of 15-lipoxygenase-1 impairs expression of HIV-1 receptors CD4 and CXCR4 in monocytic cells

The lipoxygenase pathway of immunocompetent cells has been related to HIV infection and interleukins-4 and -13 have been described as major regulators of this metabolic route. To explore whether 15-lipoxygenase1 expression might impact the responsiveness of monocytic cells for HIV we induced expression of this enzyme by two independent ways (stable transfection of U937 cells and culturing of blood monocytes in vitro in the presence of granulocyte/monocyte colony stimulating factor and interleukin 4) and assayed the cellular content of the two HIV-1 receptors CD4 and CXCR4 (CD184) by real time RT-PCR and fluorescence-activated cell sorting. Wild-type U937 cells express CD4 and CXCR4 at high levels and expression was not altered when the cells were transfected with control plasmids. In contrast, expression of these proteins was strongly reduced when the cells were stably transfected with 15-lipoxygenase1. Similar effects were observed when blood monocytes were cultured in the presence of granulocyte/monocyte colony stimulating factor and interleukin-4. Under these conditions strong overexpression of 15-lipoxygenase1 was paralleled by downregulation of CD4 and CXCR4. Since these surface proteins are essential for the binding of T-tropic HIV-1 strains expression of 15-lipoxygenase1 may confer resistance against HIV infection to monocytic cells.     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus)

A proliferation-inducing ligand (APRIL) is an important member of the tumor necrosis factor (TNF) superfamily. In the present study, a novel cDNA was isolated from the spleen of goat by RT-PCR and designated as goat APRIL (gAPRIL). The open reading frame (ORF) of this cDNA covered 753 bp, encoding a protein of 250 amino acids. Sequence comparison showed that gAPRIL contains a predicted transmembrane domain, a putative furin protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF gene in mammals. The predicted three dimensional (3D) structure of soluble part of the gAPRIL (gsAPRIL) monomer analyzed by comparative protein modeling revealed that it is very similar to its counterparts. Real-time PCR analysis revealed that gAPRIL was constitutively expressed in various tissues. Recombinant gsAPRIL fused with NusA tag was efficiently produced in Escherichia coli BL21 (DE3) and then analyzed by the SDS-PAGE as well as western blot. Laser scanning confocal microscopy analysis showed gsAPRIL could bind to its receptors. In vitro, the MTT and flow cytometric methods revealed that purified gsAPRIL protein was not only able to promote survival/proliferation of goat splenocytes, but also able to stimulate survival/proliferation of mouse B cells. These results indicated that gAPRIL plays an important role in survival/proliferation of goat splenocytes and provided a basis for investigating its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and as an immunotherapeutic in goats.