The third intracellular loop of D1 and D5 dopaminergic receptors dictates their subtype-specific PKC-induced sensitization and desensitization in a receptor conformation-dependent manner

We previously showed that phorbol-12-myristate-13-acetate (PMA) mediates a robust PKC-dependent sensitization and desensitization of the highly homologous human Gs protein and adenylyl cyclase (AC)-linked D1 (hD1R) and D5 (hD5R) dopaminergic receptors, respectively. Here, we demonstrate using forskolin-mediated AC stimulation that PMA-mediated hD1R sensitization and hD5R desensitization is not associated with changes in AC activity. We next employed a series of chimeric hD1R and hD5R to delineate the underlying structural determinants dictating the subtype-specific regulation of human D1-like receptors by PMA. We first used chimeric receptors in which the whole terminal region (TR) spanning from the extracellular face of transmembrane domain 6 to the end of cytoplasmic tail (CT) or CT alone were exchanged between hD1R and hD5R. CT and TR swaps lead to chimeric hD1R and hD5R retaining PMA-induced sensitization and desensitization of wild type parent receptors. In striking contrast, hD1R sensitization and hD5R desensitization mediated by PMA are correspondingly switched to PMA-induced receptor desensitization and sensitization following the IL3 swap between hD1R and hD5R. Cell treatment with the PKC blocker, Gö6983, inhibits PMA-induced regulation of these chimeric receptors in a similar fashion to wild type receptors. Further studies with chimeras constructed by exchanging IL3 and TR show that PMA-induced regulation of these chimeras remains fully switched relative to their respective wild type parent receptor. Interestingly, results obtained with the exchange of IL3 and TR also reveal that the D1-like subtype-specific regulation by PMA, while fully dictated by IL3, can be modulated in a receptor conformation-dependent manner. Overall, our results strongly suggest that IL3 is the critical determinant underlying the subtype-specific regulation of human D1-like receptor responsiveness by PKC.     

Targeting Immunoliposomes to Pulmonary Type II and Tracheobroncheal Epithelial Cells

Abstract

Specific agents are necessary to target therapies to certain cell types, both to maximize benefit and to minimize damage to healthy cells. Immunoliposomes (IL) may help to target the delivery of therapeutic drugs and DNA molecules to specific cells or organs. We describe here the characterization in vitro of an immunoliposome incorporating A2R, a monoclonal antibody that recognizes a receptor for surfactant protein-A (SP-A) present on both type II pneumocytes and the epithelium of the conducting airways. A2R-immunoliposomes (A2R-IL) containing β-galactosidase as a marker were prepared by coupling derivatized A2R to liposomes made of phosphatidylcholine and cholesterol. Control IL were prepared similarly, but incorporated normal IgG instead of A2R (IgG-IL), or no Ig (Null-IL). Type II cells in primary explant cell culture were treated with either A2R-IL, IgG-IL, or null-IL. We found highly significant differences in binding between specific and nonspecific IL: 31.44% of type II cells incubated with A2R-IL showed transfer of the β-gal. Pretreatment of the type II cells with SP-A blocked SP-A receptors and reduced the uptake of A2R-IL almost to background levels. As SP-A receptor is present on the cells of the conducting airways, we tested tracheal explants for their ability to take up A2R-IL, and found that these cells, similarly, could be targeted by A2R-IL. Thus, this immunoliposome could facilitate future study and therapies directed at type II alveolar cells and/or tracheobroncheal epithelium.     

Distinct function of the cytoplasmic tail in human D1-like receptor ligand binding and coupling

To delineate the role of the cytoplasmic tail in the distinct binding and coupling properties of human dopamine D1-like receptors, chimeric receptors were generated in which the entire tail region of wild-type human D1A (or D1) and D1B (or D5) receptors was exchanged. The hD1A-D1BT, but not hD1B-D1AT, receptor expression was dramatically reduced compared with wild-type receptor expression. Swapping the cytoplasmic tail resulted in a full switch of dopamine binding affinity and constitutive activity, while dopamine potency decreased and agonist-mediated maximal activation of adenylyl cyclase increased for both chimeras. Hence, the cytoplasmic tail plays a crucial role in D1-like receptor expression, agonist binding affinity and constitutive activation but regulates in a distinct fashion the formation of D1A and D1B receptor active states upon dopamine binding.     

Delineation of the structural basis for the activation properties of the dopamine D1 receptor subtypes.

To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.     

Characterization of a recombinant herpes simplex virus 1 designed to enter cells via the IL13Ralpha2 receptor of malignant glioma cells

Malignant glioma tumor cells in situ exhibit on their surfaces the interleukin 13 (IL-13) receptor designated IL13Ralpha2. To target herpes simplex virus 1 to this receptor, we constructed a recombinant virus (R5111) in which the known heparan sulfate binding sites in glycoproteins B and C were deleted and IL-13 was inserted into both glycoproteins C and D. We also transduced a baby hamster kidney cell line lacking the known viral receptors (J1-1) and Vero cells with a plasmid encoding IL13Ralpha2. The J1-1 derivative (J-13R) cell line is susceptible to and replicates the R5111 recombinant virus but not the wild-type parent virus. We report the following. (i) Expression of IL13Ralpha2 was rapidly lost from the surface of transduced cells grown in culture. The loss appeared to be related to ligands present in fetal bovine serum in the medium. None of the malignant glioma cell lines cultivated in vitro and tested to date exhibited the IL13Ralpha2 receptor. (ii) Soluble IL-13 but not IL-4 or IL-2 blocked the replication of R5111 recombinant virus in J-13R cells. (iii) The endocytosis inhibitor PD98059 blocked the replication in J1-1 cells of a mutant lacking glycoprotein D (gD-/-) but not the replication of R5111 in the J-13R cells. We conclude that R5111 enters cells via its interaction with the IL13Ralpha2 receptor in a manner that cannot be differentiated from the interaction of wild-type virus with its receptors.     

Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

Dopamine D5 receptor agonist high affinity and constitutive activity profile conferred by carboxyl-terminal tail sequence

The mammalian dopamine D1-like receptor gene family is comprised of two members, termed D1/D1A and D5/D1B. In an attempt to define the role of the carboxyl terminal (CT) tail in the expression of D5 subtype-specific pharmacological and constitutive activity profiles, we examined a series of D5 receptor chimeras in which only the CT tail was swapped with corresponding sequences encoding human/vertebrate D1-like receptors. D5/D1(CT) or D5/D1D(CT) tail substitution mutants displayed a rank order of potency and agonist affinities virtually mimicking wild-type (wt) D1 receptors, as indexed by both ligand binding and dopamine-stimulated cAMP accumulation assays, and, similar to wt D1 receptors, did not exhibit receptor constitutive activity or responsiveness to inverse agonists. D1/D5(CT) or D1/D1D(CT) tail receptor mutants displayed agonist pharmacological and functional characteristics not significantly different from parental D1 or mutant D5/D1(CT) and D5/D1D(CT) receptors. The affinities for numerous antagonists remained essentially unchanged for all receptor chimeras relative to parental wt receptors. A series of stepwise D5-CT-tail truncation/deletion mutants identified the region encoded by amino acids 438-448 and particularly Gln(439), as necessary and sufficient for the full expression of high affinity agonist and functional D5 receptor characteristics. Site-directed mutagenesis of the highly conserved D5/D1B receptor residue Gln(439)-(Ala/Ile), converts the full-length D5 receptor to one displaying "super" D5 characteristics with expressed affinities for discriminating agonists approximately 4- to 5-fold higher than wt D5 but without any concomitant increases of agonist-independent basal cAMP accumulation or intrinsic activity. Taken together, these data suggest that, in addition to other well characterized receptor domains, the agonist pharmacological and functional signature of the D5/D1B receptor is modulated by sequence-specific motifs within the CT tail and that one conserved amino acid in this region can further regulate D5 agonist high affinity binding interactions independent of receptor constitutive activity.     

Functional Differentiation of Multiple Dopamine D1-Like Receptors by NNC 01-0012

Abstract: Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype-specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1-like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS-7 cells. Of all the compounds tested, only NNC 01-0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [³H]SCH-23390 binding with an estimated affinity (∼0.6 n M ) 20-fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01-0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 µ M )-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 µ M ) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by ∼33% relative to the full agonist dopamine (10 µ M ), an effect that was blocked by the selective D1 receptor antagonist NNC 22-0010. These data clearly suggest that the benzazepine NNC 01-0012, despite lacking the N -methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01-0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.     

Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution

We created a novel mutated form of human interleukin-13 (IL-13) in which a positively charged arginine (R) at position 112 was substituted to a negatively charged aspartic acid (D). This mutant, termed IL-13R112D, was expressed in Escherichia coli and purified to near homogeneity. IL-13R112D was found to be a potent IL-13 agonist with 5-10-fold improved binding affinity to IL-13 receptors compared with wild-type IL-13 (wtIL-13). The conclusion of IL-13 agonist activity was drawn on the basis of approximately 10-fold improved activity over wtIL-13 in several assays: (a) inhibition of CD14 expression in primary monocytes; (b) proliferation of TF-1 and B9 cell lines; and (c) activation of STAT6 in Epstein-Barr virus-immortalized B cells, primary monocytes, and THP-1 monocytic cell line. Furthermore, mutant IL-13R112D neutralized the cytotoxic activity of a chimeric fusion protein composed of wtIL-13 and a Pseudomonas exotoxin A (IL-13-PE38) approximately 10 times better than wtIL-13. Based on these results, it was concluded that IL-13R112D interacts with much stronger affinity than wtIL-13 on all cell types tested and that Arg-112 plays an important role in the interaction with its receptors (IL-13R). Thus, these results suggest that IL-13R112D may be a useful ligand for the study of IL-13 interaction with its receptors or, alternatively, in designing specific targeted agents for IL-13R-positive malignancies.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Mapping the ligand-binding site on the C5a receptor: arginine74 of C5a contacts aspartate282 of the C5a receptor.

The interaction between the anaphylatoxin C5a and its receptor involves two distinct sites. One site is formed by acidic residues at the receptor N-terminus and contributes to only ligand binding. The second site, responsible for activation, is less well defined. In this study, we demonstrate that the receptor residue D(282), near the extracellular face of transmembrane domain VII, is a component of the second ligand-binding site. Mutation of D(282) to A decreases the sensitivity of the receptor to activation by intact C5a but not by its less potent metabolite, C5adR(74), which lacks the C-terminal arginine(74). The mutation of the R(74) residue of C5a to A causes a 60-fold decrease in wild-type receptor sensitivity, but only a 2-fold decrease for the receptor mutated at D(282). In contrast, the mutation of R(74) to D makes C5a completely inactive on both wild-type and A(282) C5a receptors. The mutation of D(282) to R partly restores the response to C5a[D(74)], which is a more effective ligand than C5a at the mutant receptor. A peptide mimic of the C5a activation domain with a C-terminal R potently activates the wild type but is only a weak agonist at the mutant D(282)R-C5a receptor. Conversely, a peptide with D at the C-terminus is a more effective activator of D(282)R than wild-type C5a receptors. These data indicate that the R(74) side chain of C5a makes an interaction with receptor D(282) that is responsible for the higher potency of intact C5a versus that of C5adR(74).     

Alanine-scanning mutagenesis of alpha-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine

We documented that alpha-helices A, C, and D in human interleukin-13 (IL13) participate in interaction with its respective receptors. We hypothesized that alpha-helix D is the site II of the cytokine that binds IL13Ralpha1, a component of the normal tissue heterodimeric signaling IL13/4 receptor (IL13/4R), and that alpha-helix D independently binds a monomeric IL13Ralpha2 receptor, which is a non-signaling glioma-restricted receptor for IL13. Therefore, we alanine-scanned mutagenized helix D of IL13 to identify the residues involved in the respective receptors interaction. Recombinant muteins of IL13 were produced in Escherichia coli, and their structural integrity and identity were verified. The alanine mutants were tested in functional cellular assays, in which IL13 interaction with IL13Ralpha2 (glioma cells) or an ability to functionally stimulate IL13/4R (TF-1 cells) were examined, and also in binding assays. We found that residues 105, 106, and 109 of the d-helix of IL13 are responsible for interacting with the glioma-associated receptor. Moreover, glutamic acids at positions 92 and 110, and leucine at position 104 was found to be important for IL13/4R stimulation. Thus, alpha-helix D of IL13 is the primary site responsible for interaction with the IL13 binding proteins. We propose a model that illustrates the binding mode of IL13 with cancer-related IL13Ralpha2 and physiological IL13/4R.     

Requirement of macrophages (monocytes) for the induction of interleukin 2 receptors on B lymphocytes

The role of macrophages (monocytes) for the induction of interleukin 2 receptors (IL 2R) on human B lymphocytes was studied by a direct immunofluorescence method with the use of a fluoresceinated anti-IL 2R monoclonal antibody and a flow cytofluorometer. Highly purified B lymphocytes alone did not induce IL 2R on their surface by stimulation with Staphylococcus aureus Cowan I, anti-mu antibody, or pokeweed mitogen. However, the addition of monocytes successfully induced IL 2R on B lymphocytes stimulated with these mitogens in a dose-dependent manner. Interleukin 1 (IL 1) produced by monocytes could partially replace the accessory function of monocytes. In accordance with these results, the proliferation of B lymphocytes and the differentiation to immunoglobulin-producing cells in response to IL 2 were also dependent on monocytes or IL 1. These results suggest that the accessory function of macrophages for IL 2-induced B cell activation is primarily on the induction of IL 2R on B lymphocytes.     

STAT5 activation underlies IL7 receptor-dependent B cell development

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R(-/-) background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R(-/-) mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-x(L) expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R(-/-) mice as determined by 1) the restoration of V(H) Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.     

Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT1B and 5-HT7 receptors

Serotonin (5-HT) and 5-HT receptor agonists can modify the response of the mammalian suprachiasmatic nucleus (SCN) to light. It remains uncertain which 5-HT receptor subtypes mediate these effects. The effects of 5-HT receptor activation on optic nerve-mediated input to SCN neurons were examined using whole-cell patch-clamp recordings in horizontal slices of ventral hypothalamus from the male mouse. The hypothesis that 5-HT reduces the effect of retinohypothalamic tract (RHT) input to the SCN by acting at 5-HT1B receptors was tested first. As previously described in the hamster, a mixed 5-HT(1A/1B) receptor agonist, 1-[3-(trifluoromethyl)phenyl]-piperazine hydrochloride (TFMPP), reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by selectively stimulating the optic nerve of wild-type mice. The agonist was negligibly effective in a 5-HT1B receptor knockout mouse, suggesting minimal contribution of 5-HT1A receptors to the TFMPP-induced reduction in the amplitude of the optic nerve-evoked EPSC. We next tested the hypothesis that 5-HT also reduces RHT input to the SCN via activation of 5-HT7 receptors. The mixed 5-HT(1A/7) receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), reduced the evoked EPSC amplitude in both wild-type and 5-HT1B receptor knockout mice. This effect of 8-OH-DPAT was minimally attenuated by the selective 5-HT1A receptor antagonist WAY 100635 but was reversibly and significantly reduced in the presence of ritanserin, a mixed 5-HT(2/7) receptor antagonist. Taken together with the authors' previous ultrastructural studies of 5-HT1B receptors in the mouse SCN, these results indicate that in the mouse, 5-HT reduces RHT input to the SCN by acting at 5-HT1B receptors located on RHT terminals. Moreover, activation of 5-HT7 receptors in the mouse SCN, but not 5-HT1A receptors, also results in a reduction in the amplitude of the optic nerve-evoked EPSC. The findings indicate that 5-HT may modulate RHT glutamatergic input to the SCN through 2 or more 5-HT receptors. The likely mechanism of altered RHT glutamatergic input to SCN neurons is an alteration of photic effects on the SCN circadian oscillator.     

Contribution of the Box 1 and Box 2 motifs of cytokine receptors to Jak1 association and activation

Kinases of the Jak family (Jak1/2/3 and Tyk2) interact with the membrane proximal domain of different cytokine receptors and play a critical role in the activation of cytokine and growth factor signaling pathways. In this report we demonstrate that both the Box 1 and Box 2 motif collaborate in the association and activation of Jak1 by type I interferons. Mutational analysis of the beta chain of type I interferon receptor (IFNalphaRbetaL/IFNAR2) revealed that Box 1 plays a more significant role in activation than in the association with Jak1. On the contrary, the Box 2 motif contributes more to the association with Jak1 than to kinase activation. Additionally, the study of the Jak1 binding sites on the IL2 receptor beta (IL2Rbeta), IFNgammaRalpha/IFNGR1, and IL10Ralpha/IL10R1 chains suggests that cytokine receptors have two different kinds of interaction with Jak1. One form of interaction involves the Box 1 and the previously described Box 2 motif, which we now designate as Box 2A, characterized by the VEVI and LEVL sequences present in IFNalphaRbetaL/IFNAR2 and IL2Rbeta subunits, respectively. The second form of interaction requires a motif termed Box 2B, which is present in the IFNgammaRalpha/IFNGR1 (SILLPKS) and IL10Ralpha/IL10R1 (SVLLFKK) chains. Interestingly, Box 2B localizes close to the membrane region (8-10 amino acids from the membrane) similar to Box 1, whereas Box 2A is more distal (38-58 amino acids from the membrane).     

Phospholipase C-gamma is required for agonist-induced Ca2+ entry

We report here that PLC-gamma isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-gamma1 or a lipase-inactive mutant PLC-gamma1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-gamma1 was ineffective. RNA interference to deplete either PLC-gamma1 or PLC-gamma2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-gamma2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-gamma2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-gamma2 or a lipase-inactive PLC-gamma2 mutant. The results indicate a lipase-independent role of PLC-gamma in the physiological agonist-induced activation of Ca2+ entry.     

Interleukin 2 receptor dysfunction in mice undergoing a graft-vs-host reaction

Mice of the (C57BL/10 X B10.A)F1 combination were given a single i.v. inoculation of 3 to 4 X 10(7) B10.A spleen cells, which induces a graft-vs-host (GVH)-associated immune deficiency in F1 mice. Between 1 and 4 wk later, spleen cells from the F1 mice were tested for the expression of IL 2 receptors by flow microfluorometry, using the 7D4 rat monoclonal antibody directed against an epitope murine IL 2 receptor. A reduction in intensity of spleen cell staining with 7D4 was detected as early as 8 days after parental cell inoculation, and no IL 2 receptors were detected by 28 days after initiation of GVH. Furthermore, the loss of IL 2 receptors was correlated with abrogation of proliferative responses to concanavalin A and lipopolysaccharide, of IL 2 production, and of cytotoxic T lymphocyte responses. These observations may be relevant for our understanding of GVH reactions, of immune disorders associated with GVH, and possibly of primary and acquired immunodeficiencies in general.     

The effects of charge-neutralizing mutation D6.30N on the functions of CB1 and CB2 cannabinoid receptors

Charge-neutralizing mutation D6.30N of the human cannabinoid receptor subtype 1 (CB1) and cannabinoid receptor subtype 2 (CB2) cannabinoid receptors was made to test two hypotheses: (1) D6.30 may be crucial for the functions of CB1 and CB2 receptors. (2) D6.30 may participate in an ionic lock with R3.50 that keeps the receptors in an inactive conformation. Specific ligand binding and ligand-induced inhibition of forskolin-stimulated cAMP accumulation were observed with human embryonic kidney epithelial cell line (HEK293) cells expressing wild-type CB1 and CB2, as well as CB1D6.30N and CB2D6.30N mutant receptors. There was however a decrease in maximum response of the mutant receptors compared to their wild-type counterparts, suggesting that D6.30 is essential for full activation of both CB1 and CB2 receptors. Both CB1D6.30N and CB2D6.30N demonstrated a level of constitutive activity no greater than that of their wild-type counterparts, indicating that either D6.30 does not participate in a salt bridge with R3.50, or the salt bridge is not critical for keeping cannabinoid receptors in the inactive conformation.