Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel.

N-type calcium channels are omega-conotoxin (omega-CgTx)-sensitive, voltage-dependent ion channels involved in the control of neurotransmitter release from neurons. Multiple subtypes of voltage-dependent calcium channel complexes exist, and it is the alpha 1 subunit of the complex that forms the pore through which calcium enters the cell. The primary structures of human neuronal calcium channel alpha 1B subunits were deduced by the characterization of overlapping complementary DNAs. Two forms (alpha 1B-1 and alpha 1B-2) were identified in human neuroblastoma (IMR32) cells and in the central nervous system, but not in skeletal muscle or aorta tissues. The alpha 1B-1 subunit directs the recombinant expression of N-type calcium channel activity when it is transiently co-expressed with human neuronal beta 2 and alpha 2b subunits in mammalian HEK293 cells. The recombinant channel was irreversibly blocked by omega-CgTx but was insensitive to dihydropyridines. The alpha 1B-1 alpha 2b beta 2-transfected cells displayed a single class of saturable, high-affinity (dissociation constant = 55 pM) omega-CgTx binding sites. Co-expression of the beta 2 subunit was necessary for N-type channel activity, whereas the alpha 2b subunit appeared to modulate the expression of the channel. The heterogeneity of alpha 1B subunits, along with the heterogeneity of alpha 2 and beta subunits, is consistent with multiple, biophysically distinct N-type calcium channels.

Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype.

The primary structures of human neuronal alpha 1, alpha 2, and beta subunits of a voltage-dependent Ca2+ channel were deduced by characterizing cDNAs. The alpha 1 subunit (alpha 1D) directs the recombinant expression of a dihydropyridine-sensitive L-type Ca2+ channel when coexpressed with the beta (beta 2) and the alpha 2 (alpha 2b) subunits in Xenopus oocytes. The recombinant channel is also reversibly blocked by 10-15 microM omega-conotoxin. Expression of the alpha 1D subunit alone, or coexpression with the alpha 2b subunit, did not elicit functional Ca2+ channel activity. Thus, the beta 2 subunit appears to serve an obligatory function, whereas the alpha 2b subunit appears to play an accessory role that potentiates expression of the channel. The primary transcripts encoding the alpha 1D, alpha 2, and beta subunits are differentially processed. At least two forms of neuronal alpha 1D were identified. Different forms of alpha 2 and beta transcripts were also identified in CNS, skeletal muscle, and aorta tissues.

Characterization and solubilization of vasoactive intestinal peptide receptors from rat lung membranes.

Receptors for vasoactive intestinal peptide (VIP) were characterized in rat lung membranes by binding and covalent cross-linking of [125I]VIP using ethylene glycolbis-(succinimidylsuccinate). Binding studies indicated the presence of two classes of binding sites for VIP in rat lung membranes: 0.28 +/- 0.11 pmol/mg protein high affinity receptors (Kd = 79.2 +/- 26.4 pM) and 3.3 +/- 0.9 pmol/mg protein lower affinity receptors (Kd = 4.8 +/- 2.1 nM). Furthermore, binding of [125I]VIP to rat lung receptors was inhibited by micromolar concentrations of GTP analogs, guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), and guanylylimidodiphosphate, suggesting that VIP receptors in rat lung membranes were tightly coupled to the guanine nucleotide regulatory protein (Ns). Scatchard analysis of VIP binding in the presence of GTP gamma S revealed selective inhibition of binding to high affinity sites. A 58K band was specifically labeled when membranes covalently labeled with [125I]VIP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The apparent size of this species was not altered when sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was carried out in the absence of reducing agent. Unlabeled VIP inhibited the labeling, with an IC50 of about 1 nM. A related peptide, GH-releasing factor-(1-40)OH, exhibited a much lower binding affinity, and two unrelated peptides, insulin and atrial natriuretic factor, did not inhibit labeling of the 58K species, even at micromolar concentrations. Labeling of the 58K species was inhibited in a GTP gamma S-dependent manner, suggesting the involvement of this species in the coupling to Ns. These data collectively indicated that the 58K species was a VIP-binding unit of VIP receptors in rat lung membranes. Several nondenaturing detergents were tested for extraction of labeled receptors from the membrane; the best extraction was obtained using 1% n-octyl-beta-D-glucopyranoside.

Covalent cross-linking of growth hormone-releasing factor to pituitary receptors.

We have used a bifunctional cross-linker, disuccinimidyl suberate, to covalently attach [125I]human pancreatic GH-releasing factor (GHRF) (-1-40)OH to bovine pituitary membranes and rat anterior pituitary cells. Covalently radiolabeled membrane and cell preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. In the former case, we observed the specific labeling of a polypeptide with an apparent mol wt of 75,000 +/- 3,000. The labeling of this species was specific for GHRF, as evidenced by the fact that it was inhibited in a dose-dependent fashion with increasing concentration of unlabeled GHRF. Furthermore, the radiolabeling was inhibited in the presence of excess unlabeled GHRF analogs but not unrelated peptides such as insulin and rat GH. The size of the radiolabeled band was the same in both bovine pituitary membranes and rat anterior pituitary cells. The extent of radiolabeling was dependent on the amount of membrane or the number of cells present during the binding reaction. These observations indicate that the mol wt 75,000 species is a ligand-binding subunit of the GHRF receptor in the pituitary. Under nonreducing conditions, a species much larger than mol wt 200,000 was specifically radiolabeled, again in both bovine pituitary membranes and rat cells. This result suggests the possibility that the ligand-binding subunit might be disulfide-linked to other subunit(s) forming homo- and heterooligomers.

Expression of N-methyl-D-aspartate receptor subunit mRNAs in the human brain: striatum and globus pallidus.

N-methyl-D-aspartate receptors (NRs) play an important role in basal ganglia function. By using in situ hybridization with ribonucleotide probes, we investigated the regional and cellular distribution of NR subunit mRNA expression in the human basal ganglia: caudate nucleus, putamen, lateral globus pallidus (LGP), and medial globus pallidus (MGP). Analysis of both film autoradiograms and emulsion-dipped slides revealed distinct distribution patterns for each subunit. On film autoradiograms, the signal for NR1, NR2B, and NR2C in the striatum (STR) was higher than in globus pallidus (GP). The NR2D probe gave a stronger signal in GP than in STR. For NR2A we found a signal in all regions. Analysis of emulsion-dipped sections demonstrated that in striatal neurons, the NR2B signal was higher than in GP neurons. In GP neurons, NR2D was more abundant than in striatal neurons. Despite the relatively low signal on film for NR2C in GP, we found a slightly higher signal in GP per neuron than in STR since in the pallidal areas neurons were sparse but intensely labeled. NR1 and NR2A were more evenly distributed over neurons of STR and GP Between the different parts of STR and GP, we observed only minor differences in the expression of NRs. In MGP a subpopulation of neurons exhibiting low NR2D signals could be separated from the majority of neurons showing an intense NR2D signal. Since the physiological properties of NRs are dependent on subunit composition, these data suggest a high degree of regional specialization of NR properties in the human basal ganglia.

Expression of N-methyl-D-aspartate receptor subunit mRNAs in the human brain: hippocampus and cortex.

N-methyl-D-aspartate receptor (NR) activation in the hippocampus and neocortex plays a central role in memory and cognitive function. We analyzed the cellular expression of the five NR subunit (NR1 and NR2A-D) mRNAs in these regions with in situ hybridization and human ribonucleotide probes. Film autoradiograms demonstrated a distinct pattern of hybridization signal in the hippocampal complex and the neocortex with probes for NR1, NR2A, and NR2B mRNA. NR2C and NR2D probes yielded scattered signals without a distinct organization. At the emulsion level, the NR1 probe produced high-density hybridization signals across the hippocampal complex. NR2A mRNA was higher in dentate granule cells and pyramidal cells in CA1 and subiculum compared to hilus neurons. NR2B mRNA expression was moderate throughout, with higher expression in dentate granule cells, CA1 and CA3 pyramidal cells than in hilus neurons. In the hippocampal complex, the NR2C probe signal was not different from background in any region, whereas the NR2D probe signal resulted in low to moderate grain densities. We analyzed NR subunit mRNA expression in the prefrontal, parietal, primary visual, and motor cortices. All areas displayed strong NR1 hybridization signals. NR2A and NR2B mRNAs were expressed in cortical areas and layers. NR2C mRNA was expressed at low levels in distinct layers that differed by region and the NR2D signal was equally moderate throughout all regions. Pyramidal cells in both hippocampus and neocortex express NR1, NR2A, NR2B, and, to a lesser extent, NR2D mRNA. Interneurons or granular layer neurons and some glial cells express NR2C mRNA.

Expression of N-methyl-D-aspartate receptor subunit mRNA in the human brain: mesencephalic dopaminergic neurons.

Evidence is accumulating that glutamate-mediated excitotoxicity plays an important role in neuronal degeneration in Parkinson's disease (PD). In addition, alterations in excitatory amino acid neurotransmission in the basal ganglia contribute to the clinical manifestations of motor dysfunction. However, detailed knowledge of the anatomical distribution and subtype specificity of glutamate receptors in the dopamine neurons of human substantia nigra (SN) has been lacking. In order to test the hypothesis that selective expression of particular N-methyl-D-aspartate receptor (NR) subunit mRNA contributes to the differential vulnerability of specific neuronal populations to excitotoxic injury in PD, we have used a quantitative dual label, in situ hybridization technique with ribonucleotide probes to examine the cellular distribution of NR subunit mRNA in postmortem human mesencephalic dopaminergic neurons from subjects with no known neurological disorder. Analysis of both film autoradiograms and emulsion-dipped sections demonstrated significant labeling of nigral neurons for each NR subunit. Neuronal labeling was most intense for the NR1 and NR2D subunits, with low level labeling for the remaining subunits. In addition, we examined four subregions of the ventral mesencephalon for differential expression of NR subunit mRNA. For all NR subunits, the pars lateralis (PL) exhibited the most intense signal, while neurons of the ventral tier substantia nigra pars compacta (SNpc) failed to demonstrate a preponderance of a particular subunit. These results demonstrate that NRs are expressed to a significant degree in dopaminergic neurons of the SN and that their distribution does not correlate with the characteristic pattern of neuronal degeneration in PD.

Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha 2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.

Cloning and stable expression of the mGluR1b subtype of human metabotropic receptors and pharmacological comparison with the mGluR5a subtype.

We isolated and characterized a cDNA encoding the human metabotropic glutamate receptor subtype 1b (hmGluR1b). In situ hybridization studies in human brain regions revealed a higher distribution of mGluR1 mRNA in the dentate gyrus of the hippocampus, the substantia nigra pars compacta and the Purkinje cell layer of the cerebellum compared to other regions studied. We established stable expression of recombinant hmGluR1b in L(tk-) mouse fibroblast and Chinese hamster ovary (CHO-dhfr-) cells. In both expression systems, agonist activation of hmGluR1b stimulated inositol phosphate (InsP) formation and elevation of the cytosolic free calcium ([Ca2+]i), and both responses were blocked by (S)-MCPG. The rank order of potency for agonists was quisqualate > glutamate > (1S,3R)-ACPD in both expression systems. Comparison of the agonist profiles of hmGluR1b and hmGluR5a, both stably expressed in L(tk-) cells, indicated the same rank order of potency (quisqualate > glutamate > or = (RS)-3,5-DHPG > or = (1S,3R)-ACPD), but each of the four agonists were more potent on hmGluR5a than on hmGluR1b. In antagonist studies, (S)-MCPG inhibited the agonist-induced InsP formation and elevation of [Ca2+]i in both hmGluR1b- and hmGluR5a-expressing cells. (S)-4CPG and (S)-4C3HPG both inhibited agonist responses only in hmGluR1b-expressing cells. However, in hmGluR5a-expressing cells the antagonist activity of (S)-4CPG and (S)-4C3HPG was dependent on the agonist used in the study, since they inhibited responses to glutamate but not to quisqualate. Stable cell lines expressing specific subtypes of human mGluRs represent valuable tools for the study of the mechanism of action of mGluRs at the molecular and cellular level and as screening targets for identification of subtype-selective agonists or antagonists.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Comparative structure of human neuronal alpha 2-alpha 7 and beta 2-beta 4 nicotinic acetylcholine receptor subunits and functional expression of the alpha 2, alpha 3, alpha 4, alpha 7, beta 2, and beta 4 subunits.

cDNA clones encoding human neuronal nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, beta 2, beta 3, and beta 4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human alpha 2 and alpha 6 and full-length beta 3 and beta 4 clones have not been previously reported. Deduced amino acid sequences of the alpha 2, alpha 6, beta 3, and beta 4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (alpha 2), 87 (alpha 6), 89 (beta 3), and 84% (beta 4) identity to the corresponding rat sequences. The amino termini of the human alpha 2 and beta 3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat alpha 2 and beta 3. Recombinant receptors were expressed in Xenopus laevis oocytes injected with in vitro transcripts encoding either alpha 7 alone or alpha 2, alpha 3, or alpha 4 in pairwise combination with beta 2 or beta 4. Inward currents were elicited by the application of acetylcholine (1-100 microM) and other agonists; these responses were blocked 65-97% by application of 10 microM d-tubocurare, confirming functional expression of human nicotinic receptors.

Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states.

Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363-19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.

Profiling of trans-azetidine-2,4-dicarboxylic acid at the human metabotropic glutamate receptors mGlu1b, -2, -4a and -5a.

We have tested the two enantiomers of trans-azetidine-2,4-dicarboxylic acid, (2S,4S)-azetidine-2,4-dicarboxylic acid ((2S,4S)-ADA) and (2R,4R)-azetidine-2,4-dicarboxylic acid ((2R,4R)-ADA) for activity at the human metabotropic glutamate receptors mGlu1b, mGlu2, mGlu4a and mGlu5a expressed in mammalian cells. In Chinese hamster ovary (CHO) cells expressing human mGlu2 receptors, 500 microM (2S,4S)-ADA inhibited forskolin-stimulated cAMP accumulation by 33 +/- 3% while 100 microM (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid induced an inhibition by 66 +/- 5%. The (2R,4R)-ADA enantiomer was inactive at human mGlu2 receptors. In CHO cells expressing human mGlu4a receptors, 10 microM L-AP4 inhibited forskolin-stimulated cAMP levels by 37 +/- 4% whereas both ADA enantiomers of trans-azetidine-2,4-dicarboxylic acid (500 microM) had no such effect. In CHO cells expressing human mGlu1b receptors and L cells expressing human mGlu5a receptors, both enantiomers, applied at 500 microM or 1 mM, were ineffective in stimulating inositolmonophosphate accumulation and did not affect quisqualate-stimulated inositolmonophosphate accumulation. We conclude that (2S,4S)-azetidine-2,4-dicarboxylic acid is a weak human mGlu2 receptor agonist and that (2R,4R)-azetidine-2,4-dicarboxylic acid is inactive at human mGlu2 receptors. Trans-azetidine-2,4-dicarboxylic acid has no significant agonistic effect on human mGlu4a receptors and neither agonistic nor antagonistic effects on human mGlu1b and mGlu5a receptors.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

Solubilization of rat lung vasoactive intestinal peptide receptors in the active state. Characterization of the binding properties and comparison with membrane-bound receptors.

We demonstrate here that rat lung membrane vasoactive intestinal peptide (VIP) receptors can be extracted in the active state using digitonin. Sepharose 4B gel filtration chromatography was utilized to demonstrate the formation of specific binding complexes between 125I-VIP and solubilized receptors. A rapid soluble receptor assay was established to separate 125I-VIP-receptor complexes from free 125I-VIP, which entailed differential precipitation of the 125I-VIP-receptor complex with polyethylene glycol and bovine gamma-globulin. Using this assay, several detergents were tested for their suitability to extract active VIP receptors, and most favorable results were obtained with digitonin, as judged by specific binding of 125I-VIP to the solubilized receptors. Time course studies indicated that the binding of 125I-VIP to digitonin extract was more rapid than to rat lung membranes. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract, as in the membrane. The values for the dissociation constants (Kd) were 200 pM for Class I and 8 nM for Class II receptors while the values for binding capacity (Bmax) were 200 and 2300 fmol/mg for Class I and II sites, respectively. Although the binding parameters of the two classes were similar to those in the membrane, the pharmacological properties were different, as evidenced by the inability of rat growth hormone releasing factor, a potent VIP agonist in the membrane, to displace specifically bound 125I-VIP from solubilized receptors. The ability to solubilize active VIP receptors represents an important step toward purification of the functional protein.

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside.

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.