Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization

The classical electrophysiological method to measure the function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT(3)) receptor, a cation-permeable ligand-gated ion channel, is time-consuming and not suitable for high-throughput screening. Therefore, we have optimized the conditions for a sensitive assay suitable to measure 5-HT(3) receptor responses in cell suspension based on aequorin bioluminescence caused by Ca(2+) influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT(3) receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT(3A) receptor or the human heteromeric 5-HT(3A/B) receptor in the same subset of cells. Thus, the luminescence signal originates exclusively from transfected cells, leading to a high signal/noise ratio, a major advantage compared with fluorescence techniques using Ca(2+)-sensitive dyes. The potencies of two 5-HT(3A) receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT(3A/B) receptor were comparable to those reported using other functional methods. In conclusion, the aequorin assay described here provides a convenient and highly sensitive method for functional characterization of 5-HT(3) receptors that is well suited for high-throughput screening.     

Characterization of the NR1, NR2A, and NR2C receptor proteins.

N-Methyl-D-aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1-A, NR2A-N1, and NR2C-N1, representing N-terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1-E, NR2A-C1, and NR2C-C1, derived from C-terminal sequences of these subunits. The anti-NR1-D antibody was raised against the putative internal loop of NR1. A size of 118 kDa was found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti-NR1-D and -NR1-E, but not anti-NR1-A. With the anti-NR1-A antibody, a 125-kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively. Enzymatic N-deglycosylation generated a 97-kDa protein from NR1, a 105-kDa protein from the 125-kDa protein, a 162-kDa protein from NR2A, and a 127-kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97- and 127-kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins. The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125-kDa protein was highest in the cerebellum and cortex.     

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y_{1, 2, 4, 6, 11, 12, 13, 14}), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y₄ receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y₄ receptors were distributed widely in the rat CNS. Heavy P2Y₄ receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y₄ receptors. P2Y₄ receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y₄ receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y₄ receptors. All the neurons expressing P2Y₄ receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y₄ receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y₄ receptors and NR1.     

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.     

High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.     

Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor

Adenosine and ATP/UTP are main components of the purinergic system that modulate cellular and tissue functions via specific adenosine and P2 receptors, respectively. Here, we explored the possibility that A(1) adenosine receptor (A(1)R) and P2Y(2) receptor (P2Y(2)R) form heterodimers with novel pharmacological properties. Coimmunoprecipitation showed these receptors directly associate in A(1)R/P2Y(2)R-cotransfected HEK293T cells. Agonist binding by the A(1)R was significantly inhibited by P2Y(2)R agonists only in membranes from cotransfected cells. The functional activity of A(1)R, as indicated by the G(i/o)-mediated inhibition of adenylyl cyclase, in the cotransfected cells was attenuated by the simultaneous addition of A(1)R and P2Y(2)R agonists. The increase in intracellular Ca(2+) levels induced by P2Y(2)R activation of G(q/11) was synergistically enhanced by the simultaneous addition of an A(1)R agonist in the coexpressing cells. These results suggest that oligomerization of A(1)R and P2Y(2)R generates a unique complex in which the simultaneous activation of the two receptors induces a structural alteration that interferes signaling via G(i/o) but enhances signaling via G(q/11).     

Characterization in cultured cerebellar granule cells and in the developing rat brain of mRNA variants for the NMDA receptor 2C subunit

N-Methyl-D-aspartate (NMDA) receptors are heteromeric structures resulting from the association of at least two distantly related subunit types, NR1 and one of the four NR2 subunits (NR2A-NR2D). When associated with NR1, the NR2 subunits impose specific properties to the reconstituted NMDA receptors. Although the NR1 mRNAs are expressed in the majority of central neurons, the NR2 subunits display distinct patterns of expression in the developing and adult rat brain. The NR2C subunit is barely expressed in the rat forebrain, whereas its expression increases substantially in the granule cells in the course of cerebellar development. We have identified novel NR2C splice variants in cultured cerebellar granule cells as well as in the developing cerebellum. When compared with the prototypic NR2C mRNA, these variants carry one (NR2Cb) or two (NR2Cd) insertions or a deletion (NR2Cc) and encode putative NR2C polypeptides that terminate between the third and fourth membrane segments or between the first and second membrane segments. RT-PCR analysis and in situ hybridization show that expression of the splice variants is developmentally regulated, both in the cerebellum and in the hippocampus. Electrophysiological recordings and microfluorimetry emissions in transfected human embryonic kidney 293 cells indicate that the NR2Cb variant, when expressed in combination with NR1, does not contribute to the formation of functional receptor channels. The significance of theses findings is discussed.     

The early intracellular signaling pathway for the insulin/insulin-like growth factor receptor family in the mammalian central nervous system

Several studies support the idea that the polypeptides belonging to the family of insulin and insulin-like growth factors (IGFs) play an important role in brain development and continue to be produced in discrete areas of the adult brain. In numerous neuronal populations within the olfactory bulb, the cerebral and cerebellar cortex, the hippocampus, some diencephalic and brainstem nuclei, the spinal cord and the retina, specific insulin and IGF receptors, as well as crucial components of the intracellular receptor signaling pathway have been demonstrated. Thus, mature neurons are endowed with the cellular machinery to respond to insulin and IGF stimulation. Studies in vitro and in vivo, using normal and transgenic animals, have led to the hypothesis that, in the adult brain, IGF-I not only acts as a trophic factor, but also as a neuromodulator of some higher brain functions, such as long-term potentiation and depression. Furthermore, a trophic effect on certain neuronal populations becomes clearly evident in the ischemic brain or neurodegenerative disorders. Thus, the analysis of the early intracellular signaling pathway for the insulin/IGF receptor family in the brain is providing us with new intriguing findings on the way the mammalian brain is sculpted and operates.     

大蜡螟性肽受体基因GmelSPR克隆及表达分析

交配是昆虫繁衍的重要方式,雌性交配后会显现出一系列保护后代而产生的行为,SPR则是重要的交配行为调控受体基因。本研究根据前期转录组SPR基因数据信息,利用RT-PCR扩增出大蜡螟SPR基因的完整CDS序列,命名为GmelSPR（GenBank登录号：OR347698）。生物信息学分析显示该基因编码区全长1 263 bp,编码420个氨基酸,预测分子量48.70718 kDa,理论等电点8.70,有7个跨膜螺旋区,具有典型G蛋白偶联受体蛋白特征。系统进化树比对结果显示大蜡螟GmelSPR与螟蛾科昆虫脐橙螟Amyelois transitella、印度谷螟Plodia interpunctella置信度较高。大蜡螟不同状态、不同组织中表达分析表明,未交尾雌蛾、雄蛾和已交尾雌蛾、雄蛾GmelSPR均在头部较高表达,且在未交尾雌蛾、雄蛾中表达量均高于已交尾雌蛾、雄蛾。本研究克隆了大蜡螟SPR基因,克隆结果表明GmelSPR与螟蛾科昆虫亲缘较近;表达谱分析则显示GmelSPR可能参与大蜡螟交配行为,为进一步研究大蜡螟中SPR基因功能奠定基础。     

Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested.     

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.     

An unusual cell in the central nervous system of Hirudo medicinalis L.: a neuron with ribbons and flags

Summary A neuron (cell 151) with the ability to silence efferent activity in the roots of a leech segmental ganglion was filled with horseradish peroxidase and studied by light-and electron microscopy. The neurites of cell 151 penetrate all areas of the neuropile except for a thin ventral layer. The branching pattern of the secondary neurites is highly variable. Post-and presynaptic structures of chemical synapses with clear vesicles of 25 nm diameter were identified. Neurites are mostly wrapped in glia and run in bundles among other axons. They frequently form ribbons which are 20–40 nm thick, extend several microns away from the dendrite and are followed up to 3 m in depth. They also form flags which are 0.5 m thick, spread out 10–20 m horizontally and run up to 80 m laterally. Both structures lie adjacent to or wrap around axons of other neurons, forming a gap of 8–10 nm. Flags and ribbons are typical for glia but have not been described previously as structures of neurons. Contralateral cells 151 appose each other in the commissures with a gap of 5–10 nm. The possible functional significance of these findings is discussed with respect to electrical coupling and to reception of strain.     

Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with α-bungarotoxin-binding activity

The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT_3AR), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between β-strands β1 and β2 (β1–β2), β6 and β7 (β6–β7), and β8 and β9 (β8–β9) are replaced with those of the 5HT_3AR. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops β1–β2, β6–β7, and β8–β9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT_3AR. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [¹²⁵I]-α-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the β1–β2 loop-only chimera, and the chimera containing all three 5HT_3AR loop substitutions. The remaining chimeras failed to show [¹²⁵I]-α-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops β1–β2, β6–β7, and β8–β9 are essential for the formation of a functional ligand-binding site, as evidenced by [¹²⁵I]-α-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins.     

Characterization of a rat kidney thromboxane A2 receptor: High affinity for the agonist ligand I-BOP

We have cloned a rat kidney thromboxane A2 receptor (TP) cDNA. This receptor was shown to be functional in that the thromboxane A₂ mimetics, U46619 and 1-BOP, elicited calcium transients in Xenopus oocytes and HEK293 cells expressing the TP receptor, respectively. Comparison of the affinities of the rat and human TP sites for the agonist radioligand [¹²⁵I]BOP showed that the rat TP site has about a ten-fold higher affinity for this drug (K_D = 0.5 vs. 4.4 nM) while the affinities of the two sites for other compound (U46619, I-PTH-OH) were the same. Our results are significant in that they identify a cloned TP as having a picomolar affinity for [¹²⁵I]BOP.     

DLG differentially localizes Shaker K+-channels in the central nervous system and retina of Drosophila

Subcellular localization of ion channels is crucial for the transmission of electrical signals in the nervous system. Here we show that Discs-Large (DLG), a member of the MAGUK (membrane-associated guanylate kinases) family in Drosophila, co-localizes with Shaker potassium channels (Sh Kch) in most synaptic areas of the adult brain and in the outer membrane of photoreceptors. However, DLG is absent from axonal tracts in which Sh channels are concentrated. Truncation of the C-terminal of Sh (including the PDZ binding site) disturbs its pattern of distribution in both CNS and retina, while truncation of the guanylate kinase/C-terminal domain of DLG induces ectopic localization of these channels to neuronal somata in the CNS, but does not alter the distribution of channels in photoreceptors. Immunocytochemical, membrane fractionation and detergent solubilization analysis indicate that the C-terminal of Sh Kch is required for proper trafficking to its final destination. Thus, several major conclusions emerge from this study. First, DLG plays a major role in the localization of Sh channels in the CNS and retina. Second, localization of DLG in photoreceptors but not in the CNS seems to depend on its interaction with Sh. Third, the guanylate kinase/C-terminal domain of DLG is involved in the trafficking of Shaker channels but not of DLG in the CNS. Fourth, different mechanisms for the localization of Sh Kch operate in different cell types.     

A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.     

DDR2胞外区的可溶性表达、纯化和功能检测

盘状结构域受体2（discoidin domain receptor2,DDR2)一种受体型酷氨酸蛋白激酶,其配体为纤维型胶原。胶原诱导的DDR2磷酸化可上调细胞基质金属蛋白酶1的过表达。DDR2在人体内广泛分布并与肿瘤的转移相关。为进一步研究DDR2在肿瘤转移中的作用和DDR2在细胞内的信号通路,对DDR2的胞外区进行了原核融合表达并纯化可溶性部分,获得纯度约85%的纯化蛋白,竞争结合抑制实验证明,纯化的融合蛋白可以特异性阻断Ⅱ型胶原与天然DDR2受体的结合。     

The effect of Bcl‐2, YAMA,and XIAP over‐expression on apoptosis and adenovirus production in HEK293 cell line

Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over‐expression of anti‐apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large‐scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type‐5‐adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over‐expression of bcl‐2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over‐expression of pro‐caspase‐3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase‐3 dependent apoptosis, and to a lesser degree caspase‐9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over‐expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase‐mediated activation plays an important role in virus infection and maturation. Biotechnol. Bioeng. 2009; 104: 752–765 © 2009 Wiley Periodicals, Inc.     

小鼠甘丙肽1型受体在HEK293A细胞中的表达和内化

目的:克隆小鼠甘丙肽1型受体( mGalR1)基因,构建其真核表达载体,并观察该基因在HEK293A细胞中的表达和内化.方法:应用RT - PCR方法,以小鼠下丘脑RNA为模板,扩增获得甘丙肽Ⅰ型受体(mGalR1)基因并定向克隆到pEGFP - N1真核表达载体;mGalR1 - pEGFP表达载体瞬时转染HEK293A细胞,利用激光共聚焦显微技术观察mGalR1在给予甘丙肽(10-7mol/L)0、5、10、15min刺激时的内化情况.结果:克隆得到正确的mGalR1基因全长序列,其cDNA为1047bp,共编码348个氨基酸;共聚焦显微镜结果表明mGalR1主要在膜上表达,经甘丙肽刺激5～15min后受体下膜进入胞浆.结论:成功构建真核表达载体mGalR1 - pEGFP并在HEK293A中表达;在甘丙肽刺激下小鼠甘丙肽1型受体存在内化现象.