Immunoaffinity purification of membrane protein constituents of the IM-9 lymphoblast receptor for substance P

Substance P (SP) is an undecapeptide neuromediator that stimulates human T-lymphocyte function by binding to stereospecific membrane receptors. Human IM-9 cultured B-lymphoblasts express approximately 20,000 receptors per cell for [125I]SP with a Kd of 0.3 nM. [125I]SP was specifically crosslinked by disuccinimidyl suberate to IM-9 cell membrane proteins of 78, 58, and 33 kDa. An indirect immunoaffinity purification procedure has now been developed based on immunoabsorption of detergent-solubilized [125I]SP-labeled IM-9 cell membrane proteins to anti-SP antibody that was bound to an epoxide ultraffinity high-performance liquid chromatography column, followed by elution in acidic 8 M urea. The 58- and 33-kDa SP-receptor complexes were purified to apparent homogeneity by immunoaffinity chromatography and identified by autoradiography and silver staining of sodium dodecyl sulfate-polyacryl-amide gels.     

Processing of the human IM-9 lymphoblast substance P receptor. Biosynthetic and degradation studies using a monoclonal anti-receptor antibody

Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.     

Stereospecific receptors for substance P on cultured human IM-9 lymphoblasts

The neuropeptide substance P (SP), which has been demonstrated to bind specifically to human blood T lymphocytes and to stimulate their uptake of [3H]thymidine and [3H]leucine, now is shown to bind stereospecifically to cultured human lymphoblasts of the IM-9 line. The specific binding of [3H]SP by IM-9 lymphoblasts increases linearly with the concentration of IM-9 lymphoblasts, achieves a plateau after approximately 15 to 20 min at 4 degrees C and 4 to 6 min at 37 degrees C, and is rapidly reversible at both 4 degrees C and 37 degrees C. The binding of [3H]SP at steady-state conditions demonstrates a dissociation constant (KD) of 0.65 +/- 0.19 nM (mean +/- SD, n = 5) and 22,641 +/- 6143 receptors per IM-9 lymphoblast. Maximal specific binding of [3H]SP to IM-9 lymphoblasts is observed at pH 7.4 and is dependent on the presence of Mg2+, but not Ca2+, in the medium. The peptide structural determinants of the inhibition of binding of [3H]SP to IM-9 lymphoblasts by substituent peptides and homologs of SP indicate that the receptors recognize predominantly the carboxy-terminal portion of SP. The characteristics of the interaction of SP with IM-9 lymphoblasts suggests a receptor-directed mechanism by which neuropeptides may modulate specifically the contributions of lymphocytes to immunity.     

Characterization of a monoclonal antibody against the lymphoblast substance P receptor

A murine monoclonal antibody to the IM-9 lymphoblast substance P (SP) receptor has been produced which recognizes the membrane-associated proteins of the SP receptor as demonstrated by immunoprecipitation of [125I]SP affinity-labeled and [35S]methionine biosynthetically labeled IM-9 soluble membranes. SP and anti-SP receptor binding to [35S]methionine-labeled IM-9 cell proteins were directly compared by attachment of each to affinity supports. Eluants from these affinity columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed an equivalent 33-kDa protein in both cases. This protein corresponds to one of the previously described [125I]SP specifically affinity-labeled membrane-associated proteins. In addition, two-color fluorescence-activated cell sorter analysis with human peripheral blood T lymphocytes with fluorescein-SP and rhodamine-labeled antireceptor antibody revealed a distinct population of cells (20 to 30%) that were equally labeled by both the fluorescent peptide and antibody. This result indicates that the anti-SP receptor antibody recognizes an epitope of the receptor that is common to both human peripheral blood T lymphocytes and IM-9 lymphoblast cells.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay of the acetylcholine receptor by affinity labeling

An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA), and for α-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophorus electricus and Torpedo californica and that this reaction is blocked in the presence of Naja naja siamensis α-neurotoxin and of other ligands of the acetylcholine binding site. Thus, in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct α-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 μg of receptor in the presence of 100-fold as much other protein.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

Involvement of substance P and the NK-1 receptor in human pathology

The peptide substance P (SP) shows a widespread distribution in both the central and peripheral nervous systems, but it is also present in cells not belonging to the nervous system (immune cells, liver, lung, placenta, etc.). SP is located in all body fluids, such as blood, cerebrospinal fluid, breast milk, etc. i.e. it is ubiquitous in human body. After binding to the neurokinin-1 (NK-1) receptor, SP regulates many pathophysiological functions in the central nervous system, such as emotional behavior, stress, depression, anxiety, emesis, vomiting, migraine, alcohol addiction, seizures and neurodegeneration. SP has been also implicated in pain, inflammation, hepatitis, hepatotoxicity, cholestasis, pruritus, myocarditis, bronchiolitis, abortus, bacteria and viral infection (e.g., HIV infection) and it plays an important role in cancer (e.g., tumor cell proliferation, antiapoptotic effects in tumor cells, angiogenesis, migration of tumor cells for invasion, infiltration and metastasis). This means that the SP/NK-1 receptor system is involved in the molecular bases of many human pathologies. Thus, knowledge of this system is the key for a better understanding and hence a better management of many human diseases. In this review, we update the involvement of the SP/NK-1 receptor system in the physiopathology of the above-mentioned pathologies and we suggest valuable future therapeutic interventions involving the use of NK-1 receptor antagonists, particularly in the treatment of emesis, depression, cancer, neural degeneration, inflammatory bowel disease, viral infection and pruritus, in which that system is upregulated.     

Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor

The effect of 8-bromo-cAMP and forskolin on the phosphorylation state and protein kinase activity of the insulin receptor was evaluated in cultured IM-9 lymphoblasts. 8-Bromo-cAMP (1 mM) or forskolin (10 microM) enhanced the phosphorylation of the insulin receptor purified from 32P-labeled cells by affinity chromatography on wheat germ agglutinin-agarose and immunoprecipitation with monoclonal antibody. In the absence of insulin, phosphorylation of the beta subunit of the receptor was increased approximately 2-fold by raising intracellular cAMP. Phosphoamino acid analysis of the beta subunit following treatment of cells with forskolin revealed an increase in phosphoserine and phosphothreonine residues. In contrast, the insulin-stimulated phosphorylation of the receptor occurred on serine, threonine, and tyrosine residues and was diminished by prior exposure of cells to forskolin. Pulse-chase experiments indicated that forskolin did not enhance the turnover of phosphate on the receptor of cells previously exposed to insulin. Furthermore, extracts from forskolin-treated cells did not differ from control extracts in their capacity to dephosphorylate 32P-labeled receptor isolated from cells treated with insulin. The insulin-dependent tyrosine protein kinase activity of the receptor isolated from forskolin-treated cells was approximately 50% as active as the receptor isolated from either control or insulin-treated cells. This was assessed using both histone and a peptide synthesized in accordance with the deduced amino acid sequence of a potential autophosphorylation site of the human receptor (Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys) as substrates for the protein kinase reaction. These results suggest that agents that raise intracellular cAMP increase phosphorylation of the insulin receptor on serine and threonine residues, reduce insulin-mediated receptor phosphorylation on tyrosine, serine, and threonine residues, and inhibit the insulin-dependent tyrosine protein kinase activity of the receptor. Thus cAMP may attenuate insulin action by altering the state of phosphorylation of the insulin receptor.     

Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.     

Valproic acid inhibits substance P-induced activation of protein kinase C epsilon and expression of the substance P receptor

The neuropeptide substance P (SP) has been hypothesized to be involved in the etiopathology of affective disorders. This hypothesis is based on the findings that neurokinin-1-receptor antagonists have antidepressant effects in depressed patients and that SP may worsen mood. In this study, we investigated the effect of the mood-stabilizing agents valproic acid (VPA), carbamazepine, and lithium on SP-induced gene expression. As a model system, we used primary rat astrocytes and human astrocytoma cells, which both express functional SP-receptors and, upon stimulation with SP, synthesize interleukin-6 (IL-6), a cytokine which has been shown to be elevated during the acute depressive state. We found that VPA dose-dependently inhibited SP-induced IL-6 synthesis which was seen with pre-incubation periods of 30 min, 3, 7 and 14 days, whereas carbamazepine and lithium showed no inhibitory effect. The inhibitory effect of VPA was not mediated by inhibition of the stress-regulated kinases p38 and p42/44 (Erk1/2) but by inhibition of protein kinase C epsilon activation. Furthermore, VPA down-regulated the expression of the substance P receptor (neurokinin(NK)-1-receptor) as assessed by real-time PCR. Whether both mechanisms contribute to the mood-stabilizing properties of VPA has to be evaluated in further studies.     

A site-specific bifunctional protein labeling system for affinity and fluorescent analysis

Most covalent protein labeling schemes require a choice between visual and affinity properties, requiring the use of multiple fusion systems where both attributes are needed. While not disruptive at the single experiment level, this detail becomes critical when addressing high-throughput experimentation. Here we develop a uniform site-specific protein tag for use in both fluorescent and affinity screening. Covalent protein tagging with a stilbene reporter via promiscuous phosphopantetheinyltransferase (PPTase) modification enables a switchable, antibody-elicited fluorescent response in solution or on affinity resin. For demonstration purposes, VibB, a natural fusion protein harboring a carrier protein domain, was labeled with a stilbene tag through PPTase modification with a stilbene-labeled coenzyme A analogue. Analysis of the resulting stilbene-tagged VibB was accomplished by fluorescent and Western blot analysis with anti-stilbene monoclonal antibody EP2-19G2. The illustration of this method for general application to fusion protein analysis offers a dual role in assisting both solution-based fluorescent analysis and surface-based affinity detection and purification.     

The effect of insulin, serum and dexamethasone on mRNA levels for the insulin receptor in the human lymphoblastoic cell line IM-9

The effect of insulin, serum and dexamethasone on mRNA levels in the insulin receptor in the human lymphoblastoic cell line IM-9 was examined. To this end, mRNA levels were quantitated by Northern blot analysis using a labeled cDNA probe for the insulin receptor. The presence of 0.1 microM dexamethasone in the medium had a strong stimulatory effect on mRNA levels in insulin receptor, suggesting the presence of a glucocorticoid inducible enhancer element near the insulin receptor gene. Also, the nature of the serum had an effect on insulin receptor mRNA levels, as cells maintained in 10% fetal calf serum had insulin receptor mRNA levels that were 40-50% of those found in IM-9 cells maintained in 1% newborn serum. Variations in insulin receptor mRNA levels led in each situation to concordant variations in insulin binding. Insulin levels of up to 1 microM had no effect on hybridizable insulin receptor mRNA levels making an insulin-induced feed-back mechanism on gene expression or mRNA stability unlikely.     

Regulation of receptor concentration by homologous hormone. Effect of human growth hormone on its receptor in IM-9 lymphocytes.

When cultured human lymphocytes of the IM-9 line were exposed to human growth hormone (hGH) at 37 degrees, washed for 2 hours, and incubated with 125I-hGH, the binding of 125I-hGH was reduced. The magnitude of the reduction in binding was dependent on the concentration of growth hormone present as well as the duration of the exposure. As little as 2 X 10(-11) M (0.5 ng/ml) growth hormone had a discernible effect. Growth hormone at 2 X 10(-10) M (5.0 ng/ml), which is a low resting concentration of hormone in vivo and occupies about 20% of the receptors at steady state at 30 degrees, produced a 50% reduction in binding while 20 mg/ml, which occupies about 50% of the receptors under steady state conditions, produced an 80% loss of receptors. Further increases in growth hormone concentration produced little further effect on receptor loss. Thus, the loss of receptors at a given concentration of growth hormone (up to 20 ng/ml) in the preincubation at 37 degrees was greater than the occupancy produced by that concentration of growth hormone receptors under steady state conditions at 30 degrees. Analysis of the data indicated that the decrease in binding of 125I-hGH was due to a loss of receptors per cell without any change in affinity of receptor for hormone or in cell number. The concentration of insulin receptors on these cells was affected by the insulin concentration in the medium, and the concentration of growth hormone receptors was affected by growth hormone, but neither hormone had any effect on the heterologous receptors. Exposure of the cells to cycloheximide (0.1 mM) produced a progressive but smaller loss of growth hormone receptors, and the effect of cycloheximide was additive to the receptor loss induced by growth hormone, suggesting that cycloheximide inhibited synthesis of receptors while growth hormone accelerated loss of receptors. When growth hormone was removed from the medium, receptor concentrations were restored rapidly; half of the loss was restored by 6 to 8 hours and the full complement of receptors was restored by 24 hours following removal of the hormone. If the growth hormone was removed and replaced with cycloheximide, the return of the receptors was delayed until the cycloheximide was removed. Thus restoration of the receptors appeared to require the synthesis of new proteins. These data indicate that in the IM-9 lymphocytes the concentration of growth hormone receptors is very sensitive to regulation by growth hormone and also add further support to the suggestion that hormones in general actively regulate the concentration of their own receptors.     

Biological activity and enzymic degradation of substance P analogs: implications for studies of the substance P receptor.

Partial sequences of Substance P, either free or blocked at their amino terminal, have been examined for their stability towards inactivation by homogenate or particulate fractions of rat brain and for their relative potencies as smooth muscle contractors. The C-terminal hexapeptide in both the free and blocked forms displays activity comparable to that of the longer C-terminal peptides as well as to that of the native undecapeptide. The blocked peptides, however, are much more stable than their corresponding free peptides. Among the free peptides Substance P is degraded slower than the free hexa- and hepta-peptides, suggesting that the N-terminal tetrapeptide part may play a role in stabilizing the molecule. Blocked hepta- and octapeptide analogs, carrying probe properties, may be useful for studies of the Substance P receptor.     

Binding site on human immunoglobulin G for the affinity ligand HWRGWV

Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383–Asn389 (SNGQPEN) located in the C_H3 domain. Subsequent modeling has suggested a possible three‐dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. Copyright © 2009 John Wiley & Sons, Ltd.     

Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.     

Molecular cloning, structural characterization and functional expression of the human substance P receptor.

A cDNA encoding the human substance P receptor (SPR) was isolated and the primary structure of the protein was deduced by nucleotide sequence analysis. This SPR consists of 407 residues and is a member of the G-protein coupled receptor superfamily. Comparison of rat and human SPR sequences demonstrated a 94.5% identity. The receptor was expressed in a COS-7 cell line and displayed a Kd for Tyr-1-SP binding of 0.24 nM. Ligand displacement by naturally occurring tachykinin peptides was SP much greater than neurokinin A greater than neurokinin B. SP stimulation of transfected cells resulted in a rapid and transient inositol 1,4,5-trisphosphate response. RNA blot hybridization and solution hybridization demonstrated that SPR mRNA was about 4.5 Kb in size, and was expressed in IM-9 lymphoblast and U373-MG astrocytoma cells, as well as in spinal cord and lung but not in liver.