An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.     

An antibody to dopamine D2 receptor inhibits dopamine antagonist and agonist binding to dopamine D2 receptor cDNA transfected mouse fibroblast cells.

A polyclonal antibody to dopamine D2 receptor (D2-receptor) has been used to examine the immuno-inhibition in the binding of a D2 antagonist, [3H]YM09151-2 and an agonist, PPHT-fluorescein to dopamine receptor DNA transfected mouse fibroblast cells. The specific activity of the [3H]YM09151-2 binding to transfected (Ltk-RGB) cells is 4-5 fold higher than untransfected (Ltk-) cells. The antibody is able to inhibit the [3H]YM09151-2 binding to the cell membranes from Ltk-RGB cells (Bmax 110.56 +/- 5.26 and 76.20 +/- 5.18 fmoles/mg protein in the presence of preimmune and immune sera, respectively, with no change in the Kd). The flow cytometric analysis of the PPHT-fluorescein labeled Ltk- and Ltk-RGB cells indicated that ligand specific fluorescence is associated only with small Ltk-RGB cells (second peak) and autofluorescence with large cells (first peak). Preincubation of the Ltk-RGB cells with antibody, reduced the fluorescence intensity of the PPHT-fluorescein by 20-25% without changing the auto-fluorescence. These results suggest that peptide antibody recognize D2-receptor in both membranes and in intact cells and interact at or near the ligand binding site of the receptor.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Structure and functional expression of cloned rat serotonin 5HT-2 receptor.

A complementary DNA (cDNA) encoding a serotonin receptor with 51% sequence identity to the 5HT-1C subtype was isolated from a rat brain cDNA library by homology screening. Transient expression of the cloned cDNA in mammalian cells was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes from transfected cells showed high-affinity binding of the serotonin antagonists spiperone, ketanserin and mianserin, low affinity for haloperidol (a dopamine D2 receptor antagonist), 8-OH-DPAT as well as MDL-72222 and no detectable binding of [3H]serotonin. This profile is consonant with the 5HT-2 subtype of serotonin receptors. In agreement with this assignment, serotonin increased the intracellular Ca2+ concentration and activated phosphoinositide hydrolysis in transfected mammalian cells. The agonist also elicited a current flow, blocked by spiperone, in Xenopus oocytes injected with in vitro synthesized RNA containing the cloned nucleotide sequences.     

Retinoic acid receptor gamma: specific immunodetection and phosphorylation

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma 1 (hRAR-gamma 1 and mRAR-gamma 1, respectively) were used to generate anti-RAR-gamma 1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1 gamma (A1)), region F (Ab2 gamma (mF) and Ab4 gamma (hF)) and region D2 (Ab5 gamma (D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma 1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma 1 cDNAs. They all reacted with both human and mouse RAR-gamma 1 proteins, except Ab4 gamma (hF) that was specific for hRAR-gamma 1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma 1 F region were also obtained (RP gamma (mF)) and found to be specific for mouse RAR-gamma 1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma 2 isoform which differs from RAR-gamma 1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma 1 (Ab1 gamma (A1)) only reacted with the RAR-gamma 1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma 1 and gamma 2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma 1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.     

DNA immunization with the ribosomal P2beta gene of Trypanosoma cruzi fails to induce pathogenic antibodies

Patients with chronic Chagas' heart disease (cChHD) develop a strong IgG response against the C-terminal region of the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta). These antibodies have been shown to exert an in vitro chronotropic effect on cardiocytes through stimulation of the beta1-adrenergic receptor (beta1-AR). Moreover, the presence of antibodies recognizing the TcP2beta C-terminus was associated with cardiac alterations in mice immunized with the corresponding recombinant protein. Here, we demonstrate that DNA immunization could be used to modulate the specificity of the anti-TcP2beta humoral response in order to avoid the production of pathogenic antibodies. After DNA injection, we detected IgG antibodies that were directed only to internal epitopes of the TcP2beta molecule and that did not exert anti-beta1-AR functional activity, measured as an increase in intracellular cAMP levels of transfected COS-7 cells. Accordingly, DNA-immunized mice did not present electrocardiographic alterations. These data demonstrate that anti-TcP2beta antibodies elicited by DNA immunization are completely different in their specificity and functional activity from those produced during T. cruzi infection.     

The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells.

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C-terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha-subunits, G alpha q and/or G alpha 11.     

Epitope and functional specificity of monoclonal antibodies to mouse interferon-gamma: the synthetic peptide approach

Spleen cells from hamsters immunized with recombinant mouse interferon-gamma (IFN-gamma) were fused with mouse myeloma cells, resulting in the production of four anti-IFN-gamma monoclonal antibodies. Binding of 125I-IFN-gamma by these protein A-bound antibodies was specifically blocked by cold IFN-gamma. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFN-gamma, whereas a corresponding C-terminal (95-133) peptide had no effect on binding. The N-terminal specificity of these three antibodies was confirmed by their specific binding of 125I-N-terminal (1-39) peptide. One of the N-terminal specific monoclonal antibodies inhibited both antiviral and macrophage priming (for tumor cell killing) activities of IFN-gamma, whereas the other two had no effect on either biologic function. The selectivity of the inhibition of IFN-gamma function was not due to a differential ability of the N-terminal specific antibodies to bind IFN-gamma. Blocking experiments with cold IFN-gamma and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFN-gamma. An exact determination of the epitope specificity of the monoclonal antibody that inhibited IFN-gamma function could provide insight into the structural basis for the role of the N-terminal domain in the biologic function of IFN-gamma. Polyclonal antibodies to either the N-terminal or the C-terminal peptides also inhibited both the antiviral and the macrophage-priming activities of IFN-gamma. All of the antibodies that inhibited IFN-gamma function also blocked binding of IFN-gamma to membrane receptor on cells, whereas antibodies that did not block function also did not inhibit binding. The data suggest that both the N-terminal and the C-terminal domains of IFN-gamma play an important role in its antiviral and macrophage-priming functions, possibly in a cooperative manner.     

Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor alpha-subunit

Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit.     

Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation.

Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.     

Evolution and cell biology of dopamine receptors in vertebrates

Dopamine, one of main modulatory neurotransmitters of the nervous system acts on target cells through two classes of G protein-coupled receptors, D1 and D2. The two dopamine receptor classes display different structures, interact with different regulatory partners (including heterotrimeric G proteins) and, accordingly, have independent evolutionary origins. In vertebrates, each of these receptor classes comprises several subtypes, generated by two steps of gene duplications, early in vertebrate evolution. In the D1 receptor class, the D1A, D1B, D1C and D1D subtypes, and in the D2 class, the D2, D3 and D4 receptor subtypes have been conserved in most vertebrate groups. This conservation has been driven by the acquisition, by each receptor subtype, of a small number of specific properties, which were selected for adaptive purpose in vertebrates. Among these properties, affinity for dopamine, the natural ligand, intrinsic receptor activity, and agonist-induced desensitization clearly distinguish the receptor subtypes. In addition, each dopamine receptor subtype is addressed to a specific location within neuronal networks, although detailed information is lacking for several receptor subtypes. Receptors localization at diverse subcellular places in neurons may also differ from one subtype to another, resulting in different ways of regulating cell signalisation. One challenge for future research on dopamine and its receptors would be to identify the nature of the protein partners and the molecular mechanisms involved in localizing receptors to the neuronal plasma membrane. In this respect, the evolutionary approach we have undertaken suggests that, due to gene duplications, a reasonable degree of freedom exists in the tight organisation of dopamine receptors in neurons. This "evolvability" of dopamine systems has been instrumental to adapt the vertebrate species to nearly all the possible environments.     

Detailed analysis of the region 9-30 of rat follicle stimulating hormone receptor: identification of peptide 20-30 as a potential hormone binding inhibitor

The extracellular domain (ECD) of the follicle stimulating hormone receptor (FSHR) has been shown to be a major determinant of hormone selectivity. The N-terminal 9-30 region, the sequence of which is unique to FSHR, has been extensively studied earlier and has been proposed to be an FSHR neutralizing epitope. In this study antipeptide antibodies specific to the peptide 9-30 were generated and used for identifying a specific immunodominant region within it. Overlapping peptides corresponding to the regions 9-19, 15-25 and 20-30 were synthesized. The ability of the antipeptide antibodies to 9-30 of FSHR to bind to different peptides was checked. The results indicated that the antibodies mainly recognized the peptide 20-30 and not the other two overlapping peptides. Further, the effect of the peptide 20-30 on the binding of radiolabeled FSH to its receptor was monitored. This peptide showed FSH-binding inhibitory activity with an IC(50) value of 0.598 x 10(-4)M and was more effective than the peptide 9-30 itself. Binding kinetics revealed that the observed effect of the peptide 20-30 is due to mixed type of inhibitory mechanism. This is the smallest peptide from the rat FSHR sequence having ability to inhibit FSH binding to its receptor by more than 90%.     

Synthesis and characterization of selective dopamine D2 receptor antagonists

A series of indole compounds have been prepared and evaluated for affinity at D2-like dopamine receptors using stably transfected HEK cells expressing human D2, D3, or D4 dopamine receptors. These compounds share structural elements with the classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, and benperidol. The compounds that share structural elements with N-methylspiperone and benperidol bind non-selectively to the D2 and D3 dopamine receptor subtypes. However, several of the compounds structurally similar to haloperidol were found to (a) bind to the human D2 receptor subtype with nanomolar affinity, (b) be 10- to 100-fold selective for the human D2 receptor compared to the human D3 receptor, and (c) bind with low affinity to the human D4 dopamine receptor subtype. Binding at sigma (sigma) receptor subtypes, sigma1 and sigma2, were also examined and it was found that the position of the methoxy group on the indole was pivotal in both (a) D2 versus D3 receptor selectivity and (b) affinity at sigma1 receptors. Adenylyl cyclase studies indicate that our indole compounds with the greatest D2 receptor selectivity are neutral antagonists at human D2 dopamine receptor subtypes. With stably transfected HEK cells expressing human D2 (hD2-HEK), these compounds (a) have no intrinsic activity and (b) attenuated quinpirole inhibition of adenylyl cyclase. The D2 receptor selective compounds that have been identified represent unique pharmacological tools that have potential for use in studies on the relative contribution of the D2 dopamine receptor subtypes in physiological and behavioral situations where D2-like dopaminergic receptor involvement is indicated.     

Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors.

We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.     

Structure of DNA polymerase alpha-primase complexes from mammalian cells analyzed by using monoclonal antibodies

The molecular masses of two of the four DNA polymerase alpha-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase alpha-primase complexes. Neither of these antibodies reacts with DNA polymerase alpha-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecular masses of the two main peptides of DNA polymerase alpha-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Effects of reciprocal chimeras between the C-terminal portion of third intracellular loops of the human dopamine D2 and D3 receptors

The dopamine D3 receptor is a member of the G protein-coupled superfamily of receptors. However, its coupling with intracellular events is still not well understood. We have performed chimera constructions in which amino acid residues located in a region of the receptor involved in the coupling with second messengers (the C-terminal portion of the third intracellular loop) have been exchanged between dopamine D2 and D3 receptors. Chimera constructions did not modify substantially the pharmacological profiles, nor G protein coupling, as compared to their respective wild-type receptors. However, the D2 receptor chimera, containing the C-terminal portion of the third intracellular loop of the D3 receptor, has a lower potency to inhibit cyclic AMP production. The reciprocal construction generated a D3 receptor that is fully coupled to this second messenger pathway whereas, the native D3 receptor is uncoupled to this pathway in our transfected cells. These results suggest that the sequence selected is important for specific coupling characteristics shown by these two dopamine receptor homologues.     

In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.