Solubilized Receptor Preparations Research Articles

A WHO Reference Reagent for the Serum Transferrin Receptor (sTfR): international collaborative study to evaluate a recombinant soluble transferrin receptor preparation

The usefulness of serum transferrin receptor (sTfR) as a marker of iron deficiency is limited by lack of standardization of commercial immunoassays for sTfR. An international collaborative study was performed to evaluate a lyophilized preparation of recombinant soluble transferrin receptor (rsTfR) for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize immunoassays for sTfR. The concentration of pure rsTfR was determined from the A(280 nm) using the adjusted theoretical extinction coefficient and molecular weight calculated from its sequence, before dilution and lyophilization in a sTfR-depleted serum matrix. Six manufacturers and a health protection laboratory assayed the candidate Reference Reagent, coded 07/202, along with three lyophilized serum samples, using commercial assays for sTfR. Dose-response plots demonstrated acceptable overall parallelism between 07/202, manufacturers' in-house standards, and serum samples. However, there was poor agreement on the estimated (r)sTfR content of 07/202 and serum samples. Expressing the sTfR content of the serum samples relative to 07/202 markedly improved agreement between methods. Use of 07/202 would reduce inter-method variability. The preparation was established as the 1st WHO Reference Reagent for sTfR with assigned free rsTfR monomer values of 21.7 mg/L and 303 nmol/L (0.5 mL reconstitution).

Analyzing ligand and small molecule binding activity of solubilized GPCRs using biosensor technology

We used Biacore technology to measure directly the binding of natural ligands and small molecules to the chemokine receptors CXCR4 and CCR5. Both G protein-coupled receptors were solubilized from whole cell pellets and captured on antibody surfaces for analysis. Our solubilization conditions maintained high-affinity binding of chemokines SDF-1α and RANTES to CXCR4 and CCR5, respectively. Surface density- and buffer-dependent binding responses, along with binding data for a selective ligand (RCP-168), further validated the biosensor assay. In addition, we showed that it is possible to collect high-quality binding responses for the archetypal small molecule inhibitors JM-2987 and TAK-779. Finally, using our biosensor-based method, we characterized the kinetics of 19 novel small molecule inhibitors of CCR5 and showed that their affinities correlated with values determined for the membrane-associated receptor. Together, the chemokine and small molecule binding data provide evidence that the solubilized receptors maintain native binding properties. These solubilized receptor preparations could be useful reagents for biophysical studies as well as for structural analysis.

The use of TNF family ligands and receptors and agents which modify their interaction as therapeutic agents.

The earlier known TNF family cytokines have fairly wide physiological actions, mainly in inflammation and immune responses. It is now considered feasible to develop these large proteins themselves as therapeutic agents, but in addition, modular organisation of structures of biological proteins, and the identification of localised ligand-receptor interaction sites, allow rational design of smaller, preferably nonpeptide molecules which interfere with these protein:protein interactions. Neutralising anti-TNF antibodies and soluble TNF receptor preparations were shown to have striking anti-inflammatory activities in clinical studies, particularly in rheumatoid arthritis. As the TNF beta:TNFR1 co-crystal structure was the first in the family to be solved, rational drug design based on the ligand:receptor interaction sites is more advanced. Ligand mutations and a peptide sequence from TNF-alpha have given useful information regarding ligand-receptor interactions. Small peptide sequences from TNFR I which interact with the ligand have shown some activity in blocking the biological actions of TNF. The physiological activities of several recently-discovered ligands are more limited, giving possibilities for selective treatment of several diseases. For example, TRAIL can induce apoptosis in a wide range of tumour cells with little effects on normal tissues, both in vitro and in vivo. The co-crystal structure of TRAIL with one of its signalling receptors TRAILR 2 has been solved, opening the way to rational small molecule drug design. TRANCE (RANKligand) has modulatory effects on the dendritic cell:T cell interaction in immune responses. However, it plays a more major controlling role in the development of osteoclasts and their bone resorbing activity. In this way, it is a very interesting drug development target for the treatment of bone disorders such as osteoporosis. A recombinant secreted inhibitory receptor, osteoprotegerin (OPG), is in Phase 1 clinical trial for the treatment of hyper-resorptive bone diseases. However, OPG also blocks TRAIL and may not be sufficiently specific in long term therapy, but it is hoped that inhibitors of the interaction of TRANCE and its specific signalling receptor, RANK, can be rationally designed.

Immobilized glucose-6-phosphate dehydrogenase as a substrate for solubilized epidermal growth factor receptor tyrosine kinase. A convenient microtiter plate assay system.

Based on our previously reported solution assay protocol, a solid-phase assay for the tyrosine kinase activity of the epidermal growth factor receptor has been developed. Glucose-6-phosphate dehydrogenase, immobilized noncovalently on microtiter plates, was used as the substrate in the solid-phase assay. Phosphorylation of the immobilized substrate takes place in the presence of ATP and a solubilized epidermal growth factor receptor preparation. After washing off the soluble reaction mixture, the phosphotyrosine-containing dehydrogenase produced on the well surface is quantitated by an ELISA method using a polyclonal antiphosphotyrosine antibody, a second antibody conjugated with horseradish peroxidase, and finally the o-phenylenediamine reaction. The absorbance at 492 nm developed in the wells is a measure of the kinase activity of the solubilized receptor preparation. Putative inhibitors of receptor kinase can be conveniently incorporated in this assay system to test for potential inhibitory activity. This assay, being rapid and convenient, is useful in drug screening programs where a high through-put rate is required.

Chemical Modifications of Melatonin Receptors in Chicken Brain

The membrane-bound or solubilized melatonin receptors were treated with protein-modifying agents under specific conditions and then assayed for 125I-melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl (N-ethylmaleimide and p-chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1-fluoro-2,4-dinitrobenzene) inhibited 125I-melatonin binding in a dose-dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of 125I-melatonin binding to guanine nucleotides after N-ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N-acetylimidazole, and p-nitrobenzenesulfonyl fluoride also inhibited 125I-melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross-reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited 125I-melatonin binding in a dose-dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.

Characterization of a solubilized diuretic hormone receptor from the tobacco hornworm, Manduca sexta

We have developed a procedure to solubilize a diuretic hormone receptor from the Malpighian tubules (Mt) of the tobacco hornworm, Manduca sexta in an active form using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of M. sexta diuretic hormone (Mas-DH) to the solubilized receptor preparation was saturable, specific, time dependent and reversible. Analysis of the binding data by nonlinear regression analysis indicated a single binding site with a K d = 1.1 nM and a B max = 5.8 pmol/mg solubilized protein. The solubilized receptor-Mas-DH complex could be separated from unbound hormone by centrifugation on a linear sucrose gradient and by Sepharose CL-6B chromatography. A biotinylated Mas-DH in which biotin is attached via an amide linkage to the α-amino group of the N-terminal arginine has a high affinity for the solubilized receptor. The biotinylated Mas-DH can be used to prepare an avidin coupled affinity column to purify the solubilized receptor.

Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility.

Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.

Synthesis of a Hydrophilic Affinity Matrix for the Purification of the Vasoactive Intestinal Peptide Receptor

Vasoactive intestinal peptide (VIP) was assembled on a polyacrylamide gel using a combination of the Boc and Fmoc peptide synthesis strategies. Before the synthesis, the polymeric matrix functionalized with sarcosine methylester was treated with ethylenediamine in order to form primary amine reaction sites (0.3 mmol/g). Then a six-carbon spacer arm, Boc-aminocaproic acid, was coupled to the gel after activation with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent. After acidolysis of the Boc protecting group, the derivative Boc-asparaginyl (xanthenyl)-4-(oxymethyl)phenylacetic acid was introduced into the polyacrylamide resin. Leucine-27 and isoleucine-26 were incorporated into the peptide chain as Boc-protected derivatives while the subsequent amino acids were all introduced as Fmoc residues. All couplings were achieved with BOP reagent in presence of diisopropylethylamine. The synthesis proceeded easily and only asparagine-9 required a double coupling step. After completion of the VIP assemblage, the side-chain protecting groups were removed by reaction with trifluoroacetic acid containing appropriate scavengers. A sample of peptide-resin was treated with hydrofluoric acid and the quality of the synthetic VIP-COOH material, obtained after cleavage, was assessed by reverse-phase HPLC and fast atom bombardment mass spectrometry. The compatibility with aqueous solutions of the polyacrylamide resin loaded with VIP (0.15 mmol/g), as well as its ability to be utilized as an affinity matrix for VIP receptors, was demonstrated using solubilized receptor preparation made from porcine liver membrane. After a single-step affinity chromatography, no binding activity was anymore detectable in the pass-through fraction. Moreover, after elution of the bound material, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a single band with an M r of 53 kDa after either silver staining or radioiodination. The specificity of the affinity matrix was demonstrated by adding free VIP to the detergent extract prior to the loading onto the column. Under these conditions, no protein hand was detected after SDS-PAGE analysis.

3α-Hydroxy-5α-pregnan-20-one modulation of solubilized gaba/benzodiazepine receptor complex

The allosteric modulation of the progesterone metabolite 3α-hydroxy-5α-pregnan-20-one (DHP) on [ 3H]Flunitrazepam and [ 35 S]t- butylbicyclophosphorthionate (TBPS) binding was investigated on a soluble receptor preparation. Better results in the solubilization occurred by the use of the zwitterionic detergent CHAPS with the inclusion of the phospholipid asolectin: this treatment was found suitable to study the steroidal modulation on [ 3H]Flunitrazepam and [ 35S]TBPS binding. We found that DHP was able to enhance [ 3H]Flunitrazepam binding in the presence of Cl − ions, while [ 35S]TBPS binding was inhibited by DHP. Scatchard analysis of specific [ 35S]TBPS and [ 3H]Flunitrazepam binding yielded in a single straight line both in controls and in the presence of the hormone; DHP increased the apparent affinity of [ 3H]Flunitrazepam binding without altering the apparent B max value. In the case of [ 35S]TBPS, DHP decreased the apparent B max value whereas the K d value remained nearly the same.

High-affinity insulin binding to an atypical insulin-like growth factor-I receptor in human breast cancer cells.

We studied the nature of insulin receptor binding in MCF-7 breast cancer cells. In both intact cells and solubilized receptor preparations, high-affinity insulin binding was seen. However, unlabeled insulin-like growth factor-I (IGF-I) was five-fold more potent in inhibiting 125I-insulin binding than insulin itself. With monoclonal antibodies to the insulin receptor, 30% of 125I-insulin binding was inhibited. In contrast when alpha-IR3, a monoclonal antibody that recognizes typical IGF-I receptor, was employed over 60% of 125I-insulin binding was inhibited. The B29-MAB-125I-insulin photoprobe was then cross-linked to MCF-7 membranes. Cross-linking was inhibited by both unlabeled insulin and IGF-I. Further, the B29-MAB-125I-insulin photoprobe cross-linked to MCF-7 membranes was strongly immunoprecipitated by alpha-IR3. Employing sequential affinity chromatography with insulin-Affi-gel followed by insulin receptor monoclonal antibody agarose, atypical insulin binding activity was separated from insulin receptor binding activity. This atypical receptor had intrinsic tyrosine kinase activity. Both insulin and IGF-I stimulated the phosphorylation of the receptor's beta subunit. In MCF-7 cells both IGF-I and insulin stimulated [3H]thymidine incorporation; alpha-IR3 blocked all of the IGF-I effect but only 50-60% of the insulin effect. This study demonstrates in MCF-7 cells that, in addition to typical insulin and IGF-I receptors, there is another receptor that binds both insulin and IGF-I with high affinity.

Presence of various carbohydrate moieties including beta-galactose and N-acetylglucosamine residues on solubilized porcine brain neurokinin-1/substance P receptors.

Neurokinin-1 (NK-1)/substance P (SP) receptors were solubilized using 10 mM 3-[( cholamidopropyl)-dimethylammonio]-1- propanesulfate from porcine striatal membranes (solubilization yield, 80%). In solubilized preparations, [3H]SP apparently bound to a single class of high-affinity sites (KD = 0.82 +/- 0.13 nM) as in membrane homogenates. The ligand selectivity pattern observed in both membrane and solubilized receptor preparations indicated that [Sar9,Met(O2)11]SP = SP much greater than senktide = [Nle10]neurokinin A. This suggests the selective labeling of the NK-1 receptor class in both assays. Solubilized receptors were retained on agarose-coupled lectins that bind N-acetylglucosamine-galactose and beta-galactose (Ricinus communis I and Ricinus communis II), mannose (concanavalin A and lentil), and N-acetylglucosamine (wheat germ agglutinin) but not on lectins binding fucose (Lotus A) and N-acetylgalactosamine (Doli-chos biflorus A). Thus, it appears that porcine brain NK-1/SP receptors are enriched with various carbohydrate moieties, beta-galactose and N-acetylglucosamine-galactose residues being especially abundant. This situation is rather different from that in various other members of the rhodopsin seven-transmembrane receptor superfamily.

Polylysine increases the number of insulin binding sites in soluble insulin receptor preparations.

The effects of cationic polyamino acids on insulin binding to soluble insulin receptor preparations were studied. Incubation of partially or fully purified receptor preparations with polylysine (pLys) increased by several-fold the amount of [125I]insulin that remained associated with the receptor, as determined both by precipitation of receptor-insulin complexes by polyethylene glycol or by separation of the complexes from the free hormone by gel filtration. This elevation in the amount of bound insulin resulted from increased number of insulin binding sites, and could not be attributed to an increased affinity of the receptors to insulin. In fact, pLys reduced 2-3-fold the affinity of insulin binding to its receptor as determined by equilibrium binding studies, and by monitoring the rate of exchange of bound [125I]insulin with unlabeled hormone. pLys induced specific interactions between insulin and its native receptor since other basic compounds such as histone, spermidine, polymixin B, compound 48/80, lysine, and arginine failed to reproduce its effects. pLys did not interact with the free ligand, nor did it promote interactions between insulin and denatured receptor forms. Furthermore, pLys did not induce binding of insulin to other proteins present in the partially purified receptor preparations. The effects of pLys were time and dose-dependent and were proportional to the pLys chain length. The longer the chain, the greater was the effect. Enhanced insulin binding and receptor beta-subunit autophosphorylation (in the presence of insulin) exhibited a similar dependency on the chain length of pLys. pLys effects on insulin binding were associated with formation of large protein aggregates that remained trapped at the top of Sephacryl S-300 columns. These aggregates contained substantial amounts of receptor-insulin complexes. Our results suggest that pLys induces formation of receptor clusters that create de novo insulin binding sites among adjacent receptor tetramers. Alternatively, formation of receptor aggregates might facilitate insulin binding to a soluble receptor subfraction that otherwise fails to bind the hormone.

Solubilization of a functionally active platelet-activating factor receptor from rabbit platelets.

Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface.

3H]MK‐801 Binding to N‐Methyl‐d‐Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.

Reconstitution of solubilized delta-opiate receptor binding sites in lipid vesicles.

Delta-opiate receptors have been solubilized with the non-ionic bile salt detergent digitonin from NG108-15 cell membranes and reconstituted into lipid vesicles. Specific opiate binding was restored to soluble receptor preparations after supplementation with a brain lipid extract, and dilution below the effective detergent concentration. Saturable and specific opiate binding was measured for both membrane and vesicle preparations; dissociation constants (Kd) obtained from saturation isotherms of [3H]bremazocine binding were 1.3 and 4.2 nM, respectively. Relative affinity (IC50) values of ligand binding measured for subtype-selective agonists confirmed that a delta-opiate binding site interaction was recovered in vesicle preparations. Changes in agonist binding affinity noted for these experiments were explained by dissociation of the GTP-binding protein Gi from the receptor in detergent. The recovery of solubilized opiate receptors was nearly quantitative, and strictly dependent upon the total brain lipid preparation used in the reconstitution. Ligand binding was incompletely recovered after substituting pure, vesicle-forming phospholipid preparations. [3H]Bremazocine binding was also reconstituted after lectin affinity chromatography of solubilized receptor preparations, using conditions which likely effect the removal of endogenous lipid cofactors. A photoaffinity cross-linking methodology was employed to verify recovery of the delta-opiate receptor after its solubilization from membranes and reconstitution. Two membrane-associated proteins (50 and 70 kDa) were covalently tagged with an azido analog of beta-endorphin(Leu5) in cell membranes and subsequently identified by immunoblotting with antisera directed against this opioid. Labeling of the 50-kDa polypeptide was prevented by coincubating assay samples with a relative excess of (D-Pen2,5)enkephalin. This opioid binding polypeptide was also present in solubilized/reconstituted receptor preparations.

Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors

The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.

Affinity chromatography with the immobilized agonist N-0434 yields an active and highly purified preparation of the dopamine D-2 receptor from bovine striatum

Partial purification of the dopamine D-2 receptor from bovine striatum, solubilized in the presence of 1% digitonin, was obtained by chromatography on wheat germ lectin agarose. The preparation was purified approximately 10-fold. The stability of the receptor preparation was considerably improved and non-specific protein absorption on the affinity gel used later was decreased. Further purification was achieved on a column containing a D-2-selective agonist, N-0434. Approximately 90% of the receptor activity was bound to the gel and 20–40% of the activity could be eluted by pH shock. The total purification factor after one affinity chromatogtaphy step was estimated to be at least 1500. An active preparation of at least 20% purity was obtained after a second cycle of affinity chromatography. This corresponds to an enrichment of more than 5000 times compared to the solubilized receptor preparation.

A1 adenosine receptor-G protein coupling in bovine brain membranes: effects of guanine nucleotides, salt, and solubilization.

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the D-2 dopamine receptor from bovine striatum

The D-2 dopamine receptor has been purified 21500 fold from bovine striatal membranes. Solubilized receptor preparation was partially purified by affinity chromatography on a haloperidol adsorbent followed by gel filtration on a Sephacryl S-300 column. The fractions eluted from this column which contained the ligand binding activity were further chromatographed on wheat germ agglutinin conjugated to Sepharose. The resulting receptor preparation displays a major polypeptide band of an apparent molecular weight of 92 kDa, and exhibits a specific binding activity of 2490 pmol spiperone per mg protein. This purified receptor preparation can reabsorb specifically to the haloperidol affinity column indicating that the 92 kDa polypeptide represents the ligand binding unit of the D-2 dopamine receptor.

Characterization of solubilized atrial natriuretic peptide receptors from rat olfactory bulb and A10 cultured smooth muscle cells

Atrial natriuretic peptide (ANP) receptors from A10 cultured vascular smooth muscle cells (VSMC) and rat olfactory bulbs have been solubilized and then pharmacologically and biochemically compared. The dissociation constant for 125I-ANP(99-126) was 12.7 pM for the VSMC-derived receptor and 164 pM for the olfactory receptor. Competition binding between 125I-ANP(99-126) and several unlabeled ANP analogs with the soluble olfactory receptor, demonstrated a rank order potency of ANP(99-126) = ANP(103-126) much greater than ANP(103-123). However, the rank order potency of the soluble VSMC ANP receptor was ANP(99-126) = ANP(103-126) = ANP(103-123). Therefore, the olfactory ANP receptor appears to require the complete COOH-terminal sequence of ANP as compared with the VSMC ANP receptor. When the 2 soluble receptor preparations were applied to a GTP-agarose column, a portion of the olfactory ANP receptor was retained on the column and could be eluted with 5 mM GTP, while the VSMC ANP receptor did not adsorb to the column. Since the olfactory bulb ANP receptor has been shown to contain a binding component of 116 kDa, while the VSMC ANP receptor binding component is 66 kDa, these receptors appear to be similar to the 2 receptor classes described recently in which the 120 kDa receptor that binds GTP is postulated to be coupled to guanylate cyclase, while the 60 kDa receptor does not bind GTP, is not coupled to guanylate cyclase, and may possess a hormone clearance function. Taken together, these data indicate that cyclic GMP appears to be a second messenger for ANP in the brain.