Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor.

A human BK-2 bradykinin receptor was cloned from the lung fibroblast cell line CCD-16Lu. The cDNA clone encodes a 364 amino acid protein that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The predicted amino acid sequence of the human BK-2 receptor is 81% identical to the smooth muscle rat BK-2 receptor (1). Transfection of the human BK-2 receptor cDNA into COS-7 cells results in the expression of high levels of specific BK binding sites. Saturation binding analysis indicates that the human BK-2 receptor expressed in COS-7 cells binds BK with a KD of 0.13 nM. Pharmacological characterization of the expressed BK receptor is consistent with the cDNA encoding a receptor of the BK-2 subtype. The BK-2 receptor antagonist Hoe 140 (2), D-Arg0[Hyp3, Thi5, D-Tic7, Oic8]BK has a high affinity (IC50 = 65 pM) for the cloned human receptor. The tissue distribution of the human BK-2 receptor was analyzed by competitive PCR with human tissue cDNA and is similar to that determined for the BK-2 receptor in the rat.     

Transferrin receptor 2: a new molecule in iron metabolism

Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.     

Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.     

大鼠鞘内注射多巴胺受体激动剂与拮抗剂对痛及针刺镇痛的影响

在大鼠电刺激甩测痛模型上,应用鞘内注射（ｉｔ）多巴胺（ＤＡ）受体选择性激动剂与拮抗剂,分析大鼠脊髓ＤＡ受体亚型Ｄ１和Ｄ２在痛及针刺镇痛（ＡＡ）中的作用。结果显示,在正常清醒大鼠,ｉｔ Ｄ２受体选择性激动剂,Ｙ１７１５５５（ＬＹ）或Ｄ１／Ｄ２受体激动剂阿朴吗啡（ＡＰＯ）有镇痛作用（呈剂量依赖式增加）,并加强ＡＡ,而ｉｔ Ｄ１受体选择性激动剂ＳＫＦ３８３９３（ＳＫＦ）对痛及ＡＡ均无影响;ｉｔ Ｄ１受体     

Activation of virus specific CTL clones: antigen-dependent regulation of interleukin 2 receptor expression

Interleukin 2 (IL 2) is a lymphocyte-specific growth hormone, whose effect on lymphocyte proliferation is exerted through a cell surface receptor expressed on activated lymphocytes. In this report we have used monoclonal antibodies directed to the murine IL 2 receptor to examine the regulation of the IL 2 receptor expression on cloned populations of influenza virus-specific CTL. The CTL clones, which are dependent on both specific antigenic stimulation and exogenous IL 2 for continuous in vitro propagation, express high levels of the IL 2 receptor shortly after antigenic stimulation (day 2 or 3). Over the next 5 to 8 days of in vitro cultivation in IL 2-containing medium, these cloned CTL cells express decreasing levels of IL 2 receptor. Concomitant with this fall in IL 2 receptor expression, the cells become refractory to the IL 2 proliferative stimulus. The cloned cells remain refractory to IL 2 until specifically stimulated by antigen, which induces high levels of the IL 2 receptor on the cells and renders the cells sensitive to IL 2 once again. These results support the concept that IL 2 receptor expression on activated T lymphocytes is transitory and that receptor expression is endogenously regulated in the activated T lymphocytes. These results also suggest that antigen plays a primary role in regulating T lymphocyte proliferation by maintaining IL 2 receptor levels.     

The apical targeting signal of the P2Y2 receptor is located in its first extracellular loop

P2Y2 and P2Y4 receptors, which have 52% sequence identity, are both expressed at the apical membrane of Madin-Darby canine kidney cells, but the locations of their apical targeting signals are distinctly different. The targeting signal of the P2Y2 receptor is located between the N terminus and 7TM, whereas that of the P2Y4 receptor is present in its C-terminal tail. To identify the apical targeting signal in the P2Y2 receptor, regions of the P2Y2 receptor were progressively substituted with the corresponding regions of the P2Y4 receptor lacking its targeting signal. Characterization of these chimeras and subsequent mutational analysis revealed that four amino acids (Arg95, Gly96, Asp97, and Leu108) in the first extracellular loop play a major role in apical targeting of the P2Y2 receptor. Mutation of RGD to RGE had no effect on P2Y2 receptor targeting, indicating that receptor-integrin interactions are not involved in apical targeting. P2Y2 receptor mutants were localized in a similar manner in Caco-2 colon epithelial cells. This is the first identification of an extracellular protein-based targeting signal in a seven-transmembrane receptor.     

The AT2 receptor: fact, fancy and fantasy.

The angiotensin AT2 receptor subtype was recently cloned and pharmacologically characterized but its function still remains elusive and controversial. It is a member of the G-protein coupled receptor superfamily with a minimal sequence homology with the AT1 receptor, responsible for the known effect of angiotensin II. The AT2 receptor displays a totally different signaling mechanisms from the AT1 receptor and involves various phosphatases. It is expressed at low density in adult tissues but up-regulated in pathological circumstances. Clearly, the AT2 receptor has antiproliferative properties and therefore opposes the growth promoting effect linked to the AT1 receptor stimulation. It is also reported that the AT2 receptor regulates ionic fluxes, affects differentiation and nerve regeneration, has anti-angiogenic and anti-fibrotic properties and stimulates apoptosis. However, the results, although suggestive, are sometimes equivocal. Obviously, the AT2 receptor plays a role in the pathogenesis and remodeling of cardiovascular and renal diseases. A more extensive knowledge of the AT2 receptor could therefore contribute to the understanding of the clincial beneficial effects of the AT1 receptor antagonists.     

The splice variant of the V2 vasopressin receptor adopts alternative topologies

The V2 receptor gene encodes two receptor variants by alternative splicing, the canonical V2 receptor (V2a receptor) and V2b. The V2b variant has an amino acid sequence identical to that of the V2a receptor up to the sixth transmembrane domain, but the V2b sequences corresponding to the putative seventh transmembrane domain and the carboxyl terminus are different from those of the V2a receptor. Here we investigate the topology and subcellular distribution of the V2b variant. We found that, in contrast to the V2a receptor, the V2b adopted two topologies: one with six transmembrane segments with the C-terminus on the extracellular side of the membrane and another with seven transmembrane segments with the C-terminus on the intracellular side, similar to typical G-protein-coupled receptors. Furthermore, we observed that both topological isoforms oligomerized with the V2a canonical receptor. Unlike the V2a receptor, V2b did not move to the plasma membrane, but it is retained in the ER--Golgi compartments. These findings indicate that the C-terminal sequence beyond the sixth transmembrane of the V2a is required for the stabilization of the seven-transmembrane topology of the receptor and is also essential for the trafficking of the receptor to the plasma membrane.     

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.     

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.     

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

We investigated the regulatory effects of GRK2 on D₂ dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D₂ receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D₂ receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D₂ receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγ binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.     

Development of a rat parathyroid hormone 2 receptor antagonist

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine(4), tyrosine(5), tryptophan(6), histidine(7)-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.     

Binding Ca2+ to intracellular or to extracellular sites of dihydropyridine receptor of rabbit skeletal muscle discriminates between in vitro binding of Ca2+-channel agonist and antagonist

Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an insideout orientation. Digitonin-solubilized, purified dihydropyridine receptor is embedded in digitonin vesicles in an outside-out orientation. Ca2+ selectively stimulates binding of the Ca2+-channel antagonist [3H]PN200-110 to dihydropyridine receptor in the outside-out but not the inside-out orientation. The dissociation constant for binding Ca2+ to the extracellular Ca2+-specific binding site of dihydropyridine receptor is 2-3 microM. The data demonstrate that binding Ca2+ to the extracellular high-affinity Ca2+-binding site is required for binding dihydropyridines to dihydropyridine receptor. This binding is inhibited, however, by 1-10 mM concentrations of any divalent cation tested (Ba2+, Mn2+, Mg2+). Also, Ca2+ selectively stimulates binding of the Ca2+-channel agonist [3H]BayK8644 to dihydropyridine receptor in the inside-out orientation. The titration of this Ca2+ dependence indicates that the dissociation constant for binding Ca2+ to the intracellular Ca2+-specific binding site of dihydropyridine receptor is in the millimolar range. Thus, binding Ca2+-channel agonist or antagonist to dihydropyridine receptor is modulated by binding Ca2+ to different sites of the receptor. Measurements of dissociation rate constants for binding [3H]PN200-110 to dihydropyridine receptor in the presence of diltiazem, verapamil and/or Ca2+ indicate that Ca2+, like diltiazem or verapamil, is an allosteric effector of this receptor.     

Trans-inactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP

Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic beta(2) receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.     

Formation of a uniquely stable type I interferon receptor complex by interferon beta is dependent upon particular interactions between interferon beta and its receptor and independent of tyrosine phosphorylation

Human type I interferons (IFN) require two receptor chains, IFNAR1 and IFNAR2c for high affinity (pM) binding and biological activity. Our previous studies have shown that the ligand dependent assembly of the type I IFN receptor chains is not identical for all type I IFNs. IFNbeta appears unique in its ability to assemble a stable complex of receptor chains, as demonstrated by the observation that IFNAR2c co-immunoprecipitates with IFNAR1 when cells are stimulated with IFNbeta but not with IFNalpha. The characteristics of such a receptor complex are not well defined nor is it understood if differential signaling events can be mediated by variations in receptor assembly. To further characterize the factors required for formation of such a stable receptor complex we demonstrate using IFN stimulated Daudi cells that (1) IFNAR2c co-immunoprecipitates with IFNAR1 even when tyrosine phosphorylation of receptor chains is blocked with staurosporine, and (2) IFNbeta1b but not IFNalpha2, is present in the immunoprecipitated receptor complex. These results demonstrate that the unique IFNbeta induced assembly of type I IFN receptor chains is independent of receptor tyrosine phosphorylation and the recruitment of additional proteins to the receptor by such events. Furthermore, the presence of IFNbeta1b in the immunoprecipitated IFN receptor complex suggests that IFNbeta interacts and binds differently to the receptor than IFNalpha2. These results suggest that the specific assembly of type I IFN receptor chains is ligand dependent and may represent an early event which leads to the differential biological responses observed among type I IFNs.     

Intracellular third loops in AT1 and AT2 receptors determine subtype specificity

The recently cloned angiotensin II type 2 (AT2) receptor is a member of the seven transmembrane G-protein coupled receptor superfamily with a relatively low sequence homology with the angiotensin II type 1 (AT1) receptor subtype and counteracts the growth action of AT1 receptor. Intracellular third loops are known to be involved in interactions with various G proteins. We hypothesized that the intracellular third loop plays critical roles in determining the specificity of opposite functions of AT1 and AT2 receptor subtypes and examined this possibility using chimeric AT1 receptor, of which intracellular third loop is replaced with that of AT2 receptor. We transfected this chimeric receptor into PC 12 cells and observed that stimulation of this receptor inhibited extracellular signal-regulated kinase (ERK) activation and induces apoptosis, whereas the binding characteristics of this receptor remained those of ATI receptor. Taken together, these results support the notion that intracellular third loop is the critical determinant for mutually antagonistic AT1 and AT2 receptors' signaling pathways.     

The transmembrane domain and PXXP motifs of ApoE receptor 2 exclude it from carrying out clathrin-mediated endocytosis

The low density lipoprotein (LDL) receptor family comprises several proteins with similar structures including the LDL receptor and apoE receptor 2 (apoER2). The human brain expresses two major splice variants of apoER2 mRNA, one of which includes an additional exon that encodes 59 residues in the cytoplasmic domain. This exon is absent from the LDL receptor and contains three proline-rich (PXXP) motifs that may allow apoER2 to function as a signal transducer. To investigate the role of this insert, we took advantage of the well characterized low density lipoprotein receptor pathway. Chimeras comprising the ectodomain and transmembrane domain of the LDL receptor fused to the cytoplasmic domain of apoER2 lacking the PXXP-containing residues are able to mediate clathrin-dependent endocytosis of LDL as effectively as cells expressing the LDL receptor but not if the PXXP insert is present in the protein. Although expressed on the cell surface, the PXXP-containing chimeric receptor is excluded from clathrin vesicles as judged by its failure to co-localize with adaptor protein-2 possibly due to interaction with intracellular adaptors or scaffolding proteins. Chimeras with the transmembrane domain of apoER2, predicted to be longer than that of the LDL receptor by several residues, fail to mediate endocytosis of LDL or to co-localize with adaptor protein-2 regardless of the presence or absence of the PXXP insert. Thus features of apoER2 that distinguish it as a signaling receptor, rather than as an endocytosis receptor like the LDL receptor, reside in or near the transmembrane domain and in the proline-rich motifs.     

The nature of the arrestin x receptor complex determines the ultimate fate of the internalized receptor

The vast majority of G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation of an active receptor followed by arrestin binding. The arrestin x receptor complex is then internalized. Internalized receptor can be recycled back to the plasma membrane (resensitization) or targeted to lysosomes for degradation (down-regulation). The intracellular compartment where this choice is made and the molecular mechanisms involved are largely unknown. Here we used two arrestin2 mutants that bind with high affinity to phosphorylated and unphosphorylated agonist-activated beta 2-adrenergic receptor to manipulate the receptor-arrestin interface. We found that mutants support rapid internalization of beta 2-adrenergic receptor similar to wild type arrestin2. At the same time, phosphorylation-independent arrestin2 mutants facilitate receptor recycling and sharply reduce the rate of receptor loss, effectively protecting beta 2-adrenergic receptor from down-regulation even after very long (up to 24 h) agonist exposure. Phosphorylation-independent arrestin2 mutants dramatically reduce receptor phosphorylation in response to an agonist both in vitro and in cells. Interestingly, co-expression of high levels of beta-adrenergic receptor kinase restores receptor down-regulation in the presence of mutants to the levels observed with wild type arrestin2. Our data suggest that unphosphorylated receptor internalized in complex with mutant arrestins recycles faster than phosphoreceptor and is less likely to get degraded. Thus, targeted manipulation of the characteristics of an arrestin protein that binds to a G protein-coupled receptors can dramatically change receptor trafficking and its ultimate fate in a cell.