Photoaffinity labeling of dopamine D1 receptors

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.     

Photoaffinity labeling of A1-adenosine receptors

The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for [3H]N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [3H]N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity (125I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for 125I-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that 125I-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.     

Histamine and H1 -histamine receptors faster venous circulation

The study has analysed the action of histamine in the rabbit venous system and evaluated its potential role in contraction during increased venous pressure. We have found that a great variety exists in histamine sensitivity and H(1) -histamine receptor expression in various types of rabbit veins. Veins of the extremities (saphenous vein, femoral vein, axillary vein) and abdomen (common iliac vein, inferior vena cava) responded to histamine by a prominent, concentration-dependent force generation, whereas great thoracic veins (subclavian vein, superior vena cavas, intrathoracic part of inferior vena cava) and a pelvic vein (external iliac vein) exhibited slight sensitivity to exogenous histamine. The lack of reactivity to histamine was not due to increased activity of nitric oxide synthase (NOS) or heme oxygenase-1. H(1) -histamine receptor expression of veins correlated well with the histamine-induced contractions. Voltage-dependent calcium channels mediated mainly the histamine-induced force generation of saphenous vein, whereas it did not act in the inferior vena cava. In contrast, the receptor-operated channels were not involved in this response in either vein. Tyrosine phosphorylation occurred markedly in response to histamine in the saphenous vein, but not in the inferior vena cava. Histamine induced a prominent ρ kinase activation in both vessels. Protein kinase C and mitogen-activated protein kinase (MAPK) were not implicated in the histamine-induced intracellular calcium sensitization. Importantly, transient clamping of the femoral vein in animals caused a short-term constriction, which was inhibited by H(1) -histamine receptor antagonist in vivo. Furthermore, a significantly greater histamine immunopositivity was detected in veins after stretching compared to the resting state. We conclude that histamine receptor density adapts to the actual requirements of the circulation, and histamine liberated by the venous wall during increased venous pressure contributes to the contraction of vessels, providing a force for the venous return.     

Mechanism of photoaffinity labeling of P2-purinergic receptors by arylazido aminopropionyl atp in isolated guinea-pig vas deferens

Following photolysis in the presence of the isolated guinea-pig vas deferens, arylazido aminopropionyl ATP (ANAPP₃), a photoaffinity label analog of ATP, produces an irreversible and specific pharmacological antagonism of contractile responses to adenine nucleotides. Experiments were performed to determine whether the antagonism follows the photolysis-dependent formation of nitrene intermediates at occupied receptors (true photoaffinity labeling) or if the reactive intermediate is photogenerated in solution prior to diffusion to the receptor and formation of covalent bonds (pseudo-photoaffinity labeling). When present during photolysis, para-aminobenzoic acid (PABA; 10 mM), a scavenger for nitrenes generated in solution, did not prevent the antagonism of ATP-induced responses by ANAPP₃. However, the absorption spectrum of ANAPP₃ photolyzed in the presence of PABA was different from that obtained when PABA was not present. This evidence for the formation of additional photolysis products suggests that ANAPP₃ had inserted into PABA during photolysis. Thus, covalent bonds arise from true photoaffinity labeling of the receptor. An analysis of the pharmacological effects of PABA indicated that responses to ATP, KCl and acetylcholine were unaffected either in the presence of, or after a 23 min incubation, with 10 mM PABA. In contrast, PABA produced a substantial, but reversible, antagonism of histamine- and norepinephrine-induced contractions. Irradiation of tissues in the presence of 10 mM PABA produced a slight potentiation of responses to ATP. Thus, information on the mechanisms of photoaffinity labeling may be obtained from functional studies on intact tissues. However, the pharmacological effects of agents used to define these mechanisms should be evaluated as well.     

Photoaffinity labeling of alpha 1-adrenergic receptors of rat heart

The photoaffinity probe [125I]aryl azidoprazosin was used to examine structural aspects of rat left ventricular alpha 1-adrenergic receptor. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins from photoaffinity-labeled membranes revealed a specifically labeled protein of mass 77 kDa. Adrenergic drugs competed with the photoaffinity probe for binding to the receptor in a manner expected of an alpha 1-adrenergic antagonist. Because the autoradiographic pattern was unaltered by incubating labeled membranes in gel sample buffer containing high concentrations of reducing agents, the binding component of the cardiac alpha 1-adrenergic receptor appears to be a single polypeptide chain. The photoaffinity probe specifically labeled a single protein of approximately 68 kDa in membranes of cardiac myocytes prepared from rat left ventricles. The role played by sulfhydryls in receptor structure and function was also studied. Dithiothreitol (DTT) inhibited [3H]prazosin binding to left ventricular membranes and altered both the equilibrium dissociation constant and maximal number of [3H]prazosin-binding sites but not the ability of the guanine nucleotide guanyl-5'-yl imidodiphosphate to decrease agonist affinity for the receptors. When photoaffinity-labeled membranes were incubated with 40 mM DTT for 30 min at room temperature, two specifically labeled proteins of 77 and 68 kDa were identified. The DTT-induced conversion of the 77-kDa protein to 68 kDa was irreversible with washing, but the effect of DTT on [3H]prazosin binding was reversible. Both 77- and 68-kDa proteins were observed with liver membranes even in the absence of reducing agent. We suggest that the DTT-induced conversion of the 77-kDa protein to 68 kDa is due to enhancement in protease activity by the reductant. These results document that the cardiac alpha 1-adrenergic receptor is a 77-kDa protein, similar in mass to the receptor in liver and other sites. Proteolysis likely accounts for lower Mr forms of this receptor found in cardiac myocytes and in previous publications on hepatic alpha 1-receptors.     

Lentiviral siRNA silencing of sphingosine-1-phosphate receptors S1P1 and S1P2 in smooth muscle

Sphingosine-1-phosphate regulates diverse biological processes through five receptor types, S1P(1-5). Two or more S1P receptors are usually co-expressed on target cells. We have previously shown that smooth muscle cells of the gut co-express S1P(1) and S1P(2) receptors that could mediate distinct functions. In the absence of selective agonists and antagonists, we developed siRNA constructs to silence each receptor separately. The constructs were based on identical sequences in several mammalian species. A lentiviral vector-based system was used to deliver siRNA into HEK293T cells and smooth muscle cells. One S1P(1) and two S1P(2) siRNA constructs specifically inhibited ectopic expression of S1P(1) and S1P(2) receptors, respectively, as determined by immunocytochemistry and Western blot, and endogenous expression of S1P(1) and S1P(2) receptors in smooth muscle cells, as determined by RT-PCR. Measurement of PLC-beta and Rho kinase activities, which mediate initial and sustained muscle contraction, confirmed receptor silencing and showed that contraction is mediated exclusively by S1P(2) receptors.     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.     

Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.     

Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

On the purinergic system in rat duodenum: existence of P1 and P2 receptors on the smooth muscle.

In rat duodenum, in vitro, in the presence of atropine and guanethidine, ATP administration caused a tetrodotoxin-insensitive relaxation followed by a rebound contraction. A similar response was obtained also after electrical field stimulation (EFS) of non-adrenergic, non-cholinergic (NANC) nerves. alpha, beta-methylene-TP and theophylline antagonized the response to ATP, but they failed to affect the noradrenaline- and EFS-induced relaxation. These results suggest that P1 and P2 receptors are present in rat duodenum, but their activation is not responsible for the inhibitor effects due to the NANC nerves.     

Photoaffinity labeling of parathyroid hormone receptors in clonal rat osteosarcoma cells

A photoreactive derivative of a sulfur-free bovine parathyroid hormone (PTH) analogue, [Nle8,N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH), was purified from the products of the reaction of [Nle8,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NlePTH) with 4-fluoro-3-nitro-phenylazide and was used to identify binding components of the PTH receptor in clonal rat osteosarcoma cells (ROS 17/2.8). The purified analogue, NAP-NlePTH, is a fully active agonist in three different ROS 17/2.8 cell bioassays: 1) specific binding to saturable PTH receptors; 2) stimulation of cyclic AMP accumulation; and 3) inhibition of cellular alkaline phosphatase activity; this analogue gave dose response curves parallel to and 25-33% as potent as its parent molecule, NlePTH. Radioiodinated NAP-NlePTH (125I-labeled NAP-NlePTH) retained maximal receptor-binding potency. Radioligand saturation studies in intact cells showed that the Kd of PTH receptors for the photoligand was slightly less than that for 125I-labeled NlePTH (2.8 and 0.8 nM, respectively), but that the Bmax was essentially identical for both radioligands (8 fmol/10(5) cells). Photoaffinity labeling of ROS 17/2.8 cells revealed several 125I-labeled macromolecular components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One predominant 125I-labeled band, having an apparent Mr of 80,000 daltons (including Mr = 4,347 ligand; hereafter referred to as the Mr = 80,000 protein), was consistently demonstrated in both reducing and nonreducing conditions. Its labeling was completely inhibited by coincubation with NlePTH (10 nM) at 26-fold molar excess to the photoligand, but not by biologically inactive PTH fragments or unrelated hormone. Labeling of several other macromolecular components persisted in the presence of NlePTH (1 microM). Only the labeling of the Mr = 80,000 protein showed saturation kinetics for photoaffinity labeling; the dose of 125I-labeled NAP-NlePTH (0.8 nM) to half-saturate labeling of the Mr = 80,000 protein was close to the Kd (2.8 nM) of specific binding of the photoligand to receptors in intact ROS 17/2.8 cells. Pretreatment of the cells with NlePTH and dexamethasone led to the predicted proportional decrease or increase, respectively, in labeling of the Mr = 80,000 protein. Our data, using a highly purified photoactive derivative of PTH, having carefully defined chemical and biological properties, show a plasma membrane component of Mr = 80,000 in ROS 17/2.8 cells that possesses the affinity, binding capacity, and physiological characteristics of the PTH receptor.     

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.     

Molecular and cellular analysis of histamine H1 receptors on cultured smooth muscle cells

Histamine is an important mediator of immediate hypersensitivity for both animals and humans. The action of histamine on target tissues is believed to be mediated by specific cell surface receptors, especially H1 and H2 receptors for hypersensitivity and inflammatory reactions, which involve stimulation of smooth muscle contractility, alterations in vascular permeability, and modifications in the activities of macrophages and lymphocytes. Although the nature of histamine receptors in the brain and peripheral tissues has been studied extensively by many laboratories, the molecular mechanism of histamine receptor-mediated reactions is not fully understood, mainly because histamine receptors are incompletely characterized from the biochemical point of view. In previous studies, we have found that the cultured smooth muscle cell line DDT1MF-2, derived from hamster vas deferens, expresses low-affinity histamine H1 receptors and responds biochemically and functionally to H1-specific stimulation (Mitsuhashi and Payan, J Cell Physiol 134:367, 1988). This cell line provides a model for analyzing the biochemical responses of H1 receptor-mediated reactions in peripheral tissues. In this review, we summarized our recent progress in the study of low-affinity H1 receptors on DDT1MF-2 cells.     

MAPKK-dependent growth factor-induced upregulation of P2Y2 receptors in vascular smooth muscle cells.

The ATP- and UTP-sensitive P2Y2 receptor which mediates both contractile and mitogenic effects has recently been shown to be upregulated in the synthetic phenotype of the vascular smooth muscle cell (VSMC). Using a competitive RT-PCR we demonstrate that the P2Y2 receptor mRNA is increased by fetal calf serum and other growth factors in a MAPKK-dependent way. This was confirmed at the functional level by examining UTP-stimulated release of intracellular Ca2+. Furthermore, the P2Y2 receptor mRNA is positively autoregulated by ATP and the mRNA is rapidly degraded with only 26% remaining after 1 h in the presence of actinomycin D. Our results indicate growth factor regulation and rapid turnover of the P2Y2 receptor mRNA, which may be of importance in atherosclerosis and neointima formation after balloon angioplasty.