MATERIALS AND METHODS Expression and Purification of Proteins-The vector for expression of FIP-CB SM is similar to the one described by Mitra et al. (5). The coding sequences for the BGFP (6) and RGFP (7) domains were produced by amplifying the GFP-encoding sequences in the BioBlue™ and BioYellow™ vectors obtained from PharMingen, Inc. (San Diego, CA). The vector pETIC, encoding a fluorescent indicator protein (FIP) control consisting of RGFP and BGFP domains joined by the linker sequence, GTSSGSSTGA, was generated first. The RGFP domain in pETIC is fused to the His 6 -tag/thrombin/S-tag/enterokinase leader sequence derived from pET30a (Novagen, Inc., Madison, WI). The C terminus of the BGFP domain is fused to an additional His 6 sequence, also derived from pET30a. The vector pETIC-1 encodes FIP-CB SM , which is identical to the FIP control, except that the linker has the sequence: GTSSRRKWNKTGHAVRAIGRLSSTGA. Boldface type denotes the CaM-binding sequence from avian smooth muscle myosin light chain kinase (8).For expression, pETIC and pETIC-1 were transformed into Escherichia coli strain BL21(DE3). Cells containing pETIC or pETIC-1 were grown at 23°C to an A 600 of 0.6 -0.8, and protein expression was induced by addition of isopropyl-1-thio-␤-D-galactopyranoside to 0.5 mM. After incubating at 23°C for ϳ40 h, cells were harvested. Control FIP and FIP-CB SM were purified using His 6 affinity chromatography essentially as described by Mitra et al. (5). Vertebrate CaM expressed in E. coli was purified as described previously (9). The concentrations of control FIP and FIP-CB SM were determined using an ⑀ 490 of 89 mM Ϫ1 cm Ϫ1 . Concentrations of FIP-CB SM stock solutions were verified by titration with a standard CaM solution.In Vitro Measurements of FIP-CB SM Fluorescence-Fluorescence measurements were performed using a Photon Technology International (Monmouth Junction, NJ) QuantaMaster™ photon counting spectrofluorometer. Reaction volumes (3 ml) were incubated at 30°C in a stirred cuvette. Excitation and emission slit widths were 5 nm. An excitation wavelength of 380 nm was used for in vitro measurements of FIP fluorescence. For most experiments, a buffer containing 25 mM Tris, 0.1 M NaCl, and 300 M CaCl 2 , pH 7.5, was used. For experiments in which the free Ca 2ϩ ion concentration was varied, a buffer containing 50 mM Tris, 0.1 M NaCl, 0.5 mM MgCl 2 , and 3 mM 1,2-bis(2-amino-5,5Ј-dibromophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, pH 7.5, was used. Aliquots of standard CaCl 2 solutions were added to achieve various levels of free Ca 2ϩ ion, which were calculated using the MaxChelator program (10). The FIP-CB SM fluorescence emission spectrum is essentially independent of the pH between 7.0 and 8.0, either in the presence or absence of bound (Ca 2ϩ ) 4 -CaM.

Morphine and other opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC ␤3-deficient mice, we have established a strong link between PLC and opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC ␤3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED 50 value for morphine in producing antinociception. The reduced ED 50 value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B max and K d values for , , and ␦ opioid receptors between wild-type and PLC ␤3-null mice. We also found that opioid regulation of voltage-sensitive Ca 2؉ channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific agonist [D-Ala 2 ,(Me)Phe 4 , Gly(ol) 5 ]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC ␤3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC ␤3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca 2؉ current reduction. These data demonstrate that PLC ␤3 constitutes a significant pathway involved in negative modulation of opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.Morphine and other opioids are widely used analgesics. Three opioid receptors, , ␦, and , have been cloned and characterized both functionally and by radioligand binding (1-4). These receptors couple to G i and G o proteins and, through ␣ and ␤␥ subunits, regulate a number of signaling pathways. These regulated pathways include inhibition of adenylyl cyclase activity (5, 6), activation of inwardly rectifying K ϩ channels (7,8), and inhibition of voltage-activated Ca 2ϩ channels, predominantly of the N and P͞Q types (9, 10). Recent work with cell lines has demonstrated that opioid receptors also activate phospholipase C (PLC; 11-17). Moreover, several physiological studies have implicated PLC-linked pathways in a diverse range of opioid-modulated events, such as pain regulation (18), response to brain injury (19), and opioid withdrawal (20). However, despite these data suggesting an important role for PLC-mediated pathways in opioid signal transduction, there is little direct evidence supporting a role of PLC in opioid responses.PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce two important second messengers, inositol triphosphate and diacylglycerol (21). The former increases release of Ca 2ϩ from intracellular stores, and the latter activates protein kinase C (PKC). Molecular cloning and biochemical characterizations have revealed four ...

Phospholipase C ␤2 (PLC ␤2) is activated by G protein ␤␥ subunits and calcium. The enzyme is soluble and its substrate, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), is present in phospholipid membranes. A potential mechanism for regulation of this enzyme is through influencing the equilibrium association of the enzyme with membrane surfaces. In this paper we describe a fluorescence resonance energy transfer (FRET) method for measuring the association of PLC ␤2 with phospholipid bilayers. The method allows equilibrium measurements to be made under a variety of conditions, including those that support enzymatic activity and ability to be regulated by G proteins. Using this method it was found that PLC ␤2 bound to vesicles containing anionic lipids and demonstrated a selective and unique interaction with PIP 2 -containing vesicles. The FRET data were corroborated with a centrifugation based method for estimating the affinity of PLC ␤2 for vesicles. Apparently different modes of association of PLC ␤2 with vesicles of different composition can be distinguished based on alterations in resonance energy transfer efficiency. Association of PLC ␤2 with PIP 2 vesicles requires an intact lipid bilayer, is blocked by neomycin, and is not affected by D-myo-inositol 1,4,5-trisphosphate (D-IP 3 ). G protein ␤␥ subunits do not alter the affinity of PLC ␤2 for lipid bilayers and at the PIP 2 concentrations used to measure ␤␥-dependent stimulation of PLC activity, the majority of the PLC ␤2 is already associated with the vesicle surface. Furthermore, under conditions where ␤␥ subunits strongly activate PLC activity, the extent of association with vesicles is unaffected by ␤␥ subunits or calcium. These results indicate that activation of PLC ␤2 by G protein ␤␥ subunits or Ca 2؉ in vitro does not involve translocation to the vesicle surface. Enzymes of the phospholipase C ␤ (PLC ␤)1 class hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in response to activation of G protein-coupled receptors (1). Many receptor-coupled responses that are insensitive to treatment with the toxin from Bordatella pertussis are mediated by ␣ subunits of the G q family members (2-4), while responses inhibited by this toxin are thought to be via the ␤␥ subunits from G i proteins. Both of these G proteins can activate PLC ␤ through direct proteinprotein interactions as has been demonstrated in reconstitution assays with purified proteins (1, 5-8) G protein ␤␥ subunits have been implicated as primary signal transducers in wide variety of signal transduction pathways. ␤␥ subunits have been shown to directly activate some isoforms of adenylyl cyclase and inhibit others (9), activate ␤-adrenergic receptor kinase (10), muscarinic receptor kinase (11), cardiac K ϩ channels (12), and phosphatidylinositol 3-kinase (13,14). ␤␥ subunits mediate cell cycle arrest via the mating pathway in the yeast Saccharomyces cerevisiae (15), but the effector involved has not been identified. Other work suggests that ␤␥ subunits can lead to activation of Ras and mitogen-...

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These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.