Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Multimeric structure of the tumor necrosis factor receptor of HeLa cells

The tumor necrosis factor (TNF) receptor of HeLa cells was solubilized in Triton X-100 and characterized by gel filtration, affinity labeling, and ligand blotting studies. Receptors solubilized with Triton X-100 eluted in gel filtration as a major peak of Mr = 330,000 and retained high affinity binding (KD = 0.25 nM). Affinity labeling of soluble receptor/125I-TNF complexes using the reversible, bifunctional bis[2-(succinimidooxycarbonyl-oxy)ethyl] sulfone resulted in the formation of cross-linked species of Mr = 310,000, 150,000-175,000, 95,000, and 75,000. The formation of these complexes was competitively inhibited by unlabeled TNF. Partial reversal of cross-linking in these complexes and their analysis by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 125I-TNF dimers cleaved from the 95,000 band and 125I-TNF monomer cleaved from the 75,000 band, providing evidence for a Mr approximately 60,000 subunit. In addition, the 95,000 and 75,000 bands were resolved as components of larger complexes (Mr = 150,000-175,000), which presumably contain two receptor subunits. The Mr 95,000 and 75,000 bands were also released from the Mr 310,000 complex by reduction with dithiothreitol, suggesting a role for disulfide bond stabilization. To investigate the association of the putative receptor subunits, Triton X-100 extracts from HeLa membranes were fractionated by SDS-PAGE without reduction and transferred electrophoretically to nylon membranes for TNF binding assays. Only two bands of Mr = 60,000 and 70,000 specifically bound TNF, and higher Mr binding activity was not observed. These results indicate that TNF receptors in HeLa cells are high molecular weight complexes containing Mr = 60,000 and 70,000 subunits each capable of binding TNF and that the complexes are primarily stabilized by non-covalent, hydrophobic interactions.     

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.     

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.     

Photoaffinity labeling of ANF receptor in cultured brain neurones

A monoiodo derivative of rat atrial natriuretic factor (rANF) was shown to specifically bind to rat brain neurones in culture with low binding site capacity (10-20 fmoles per mg of protein) and high affinity (Kd = 50-100 pM). Several analogs of both rat and human ANF competed with 125I-rANF. No change in the number of binding sites was detected upon morphological differentiation of neurones in vitro. Finally a photoreactive derivative of 125I-rANF was prepared and photoaffinity labeling experiments carried out on cultured neurones. After reduction of disulfide bridges, a single band of Mr 60,000 was specifically labeled whereas without reduction, two labeled components of Mr 60,000 and 117,000 were detected.     

Solubilization and molecular characterization of active galanin receptors from rat brain.

Galanin receptors were solubilized from rat brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of 125I-galanin to the soluble fraction was time- and temperature-dependent, saturable, and reversible. Scatchard analysis of binding data indicated that the soluble extract contained a single class of galanin binding sites with a Kd of 0.8 nM and a Bmax of 26 fmol/mg of protein. Unlabeled galanin and its fragments galanin(2-29) and galanin(1-15) antagonized the binding of 125I-galanin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane receptors. Galanin(3-29) was found inactive. Binding of 125I-galanin to CHAPS extracts was inhibited by guanine nucleotides with the following rank order of potency: GMP-P-(NH)P greater than GTP greater than GDP. Molecular analysis of the soluble galanin receptor by covalent cross-linking of 125I-galanin to CHAPS extracts using disuccinimidyl tartrate and further identification on SDS-PAGE indicated that the soluble galanin binding site behaves as a protein of Mr 54,000. After incubation of CHAPS extracts with 125I-galanin, gel filtration on Sephacryl S-300 followed by ultracentrifugation on sucrose density gradient revealed a binding component with the following hydrodynamic parameters: Stokes radius, 5 nm; s20,w, 4.5 S; Mr, 98,000; frictional ratio, 1.6. GMP-P(NH)P treatment of CHAPS extracts gave rise to a molecular form with the following characteristics: Stokes radius, 4 nm; s20,w, 3.3 S; Mr, 57,000; frictional ratio, 1.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characteristics of receptors for atrial natriuretic factor

Specific, high-affinity receptors for atrial natriuretic factor (ANF) have been identified on membranes from a variety of tissues and cultured cells. By affinity labeling procedures, radioactivity from 125I-labeled ANF was specifically incorporated into three different polypeptides of ca. 120,000, 70,000, and 60,000 daltons, which may represent the binding subunits of ANF receptors. These polypeptides were present in varying amounts in different target tissues. In rat adrenal membranes, the 120,000- and 70,000-dalton peptides were specifically labeled whereas in A10 rat smooth muscle cells, only the 60,000-dalton peptide was labeled. Membranes from rat kidney and rabbit aorta contain all three peptides. Gel filtration chromatography of solubilized receptors suggested that intact ANF receptors are large molecular complexes with apparent molecular masses in the range of 250,000-350,000 daltons. The differential labeling pattern observed with the various tissues suggested that there might be at least two different receptors composed of unique ANF-binding polypeptides.     

Characterization of vasoactive intestinal peptide (VIP) receptors in mammalian lung

125I-VIP bound specifically to sites on human, rat, guinea pig, and rabbit lung membranes with a dissociation constant (KD) of 60-200 pM and binding site maxima of 200-800 fmol/mg of protein. The presence of a second lower affinity site was detected but not investigated further. High affinity 125I-VIP binding was reversible and displaced by structurally related peptides with an order of potency: VIP greater than rGRF greater than PHI greater than hGRF greater than secretin = Ac Tyr1 D Phe2 GRF. 125I-VIP has been covalently incorporated into lung membranes using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrilamide gel electrophoresis of labeled human, rat, and rabbit lung membranes revealed major 125I-VIP-receptor complexes of: Mr = 65,000, 56,000, and 64,000 daltons, respectively. Guinea pig lung membranes exhibited two 125I-VIP-receptor complexes of Mr = 66,000 and 60,000 daltons. This labeling pattern probably reflects the presence of differentially glycosylated forms of the same receptor since treatment with neuroaminidase resulted in a single homogeneous band (Mr = 57,000 daltons). Soluble covalently labeled VIP receptors from guinea pig and human lung bound to and were specifically eluted from agarose-linked wheat germ agglutinin columns. Our studies indicate that mammalian lung VIP receptors are glycoproteins containing terminal sialic acid residues.     

Delta 6- and delta 12-desaturase activities and phosphatidic acid formation in microsomal preparations from the developing cotyledons of common borage (Borago officinalis).

The somatostatin receptors on rat pancreatic acinar membranes were demonstrated by use of a radioiodinated (125I-) analogue of somatostatin (SMS 204-090 or [Tyr3]SMS). The tracer was found to bind to the receptor with a Kd of 58 pM. The number of sites detected by this tracer (4.7 pmol/mg of protein) was 5-10 times higher than the number of sites previously found with other tracers. Since the level of non-specific binding was also very low as compared with findings with other tracers, 125I-204-090 might be of interest in future attempts to characterize the somatostatin receptors in the pancreas. The prelabelled membranes were solubilized with 1% CHAPS, and the solubilized complexes were found to adsorb to wheat-germ-agglutinin-coupled agarose, from which they could be eluted with 4 mM-triacetylchitotriose. The complexes within this eluate were shown by gel filtration on Trisacryl GF-2000 to have an Mr of about 400,000. The dissociation of the complexes was augmented both within the membranes as well as in the solubilized state by incubation with the GTP analogue guanosine 5'-[gamma-thio]triphosphate, indicating that the complexes are probably functionally linked to a guanine-nucleotide-binding regulatory protein. After SDS/slab-gel electrophoresis and autoradiography of cross-linked complexes after treatment with the heterobifunctional reagent N-5-azido-2-nitrobenzoyloxysuccinimide, a broad band occurred at approximately Mr 90,000 both in the membranes and in the eluates of complexes after lectin-adsorption chromatography. We conclude that the augmentation of the number of detectable sites for binding of somatostatin, as well as the very low level of non-specific binding obtained by the use of 125I-[Tyr3]SMS as tracer, has made it possible for us to demonstrate the solubilization of the somatostatin receptor in conjunction with its ligand and a GTP-binding regulatory protein, and we have succeeded in cross-linking 125I-[Tyr3]SMS to a binding subunit of Mr 90,000 in the membranes and in demonstrating the presence of the same labelled binding subunit within complexes solubilized and chromatographed on a lectin column before cross-linking.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.     

The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.     

Solubilization and identification of human placental endothelin receptor

Endothelin-1 (ET-1) receptor was identified on the membranes from human placenta and 66% of original binding activity in the membranes was solubilized with 0.75% (w/v) CHAPS. Binding studies of the solubilized membranes using 125I-ET-1 indicated the presence of a single class of high-affinity binding sites with an apparent Kd of 760 pM and a Bmax of 1.8 pmol/mg of protein. The binding was inhibited by addition of unlabeled ET-1 and ET-3 in dose dependent manner. The Ki values of solubilized membranes were 84 pM for ET-1 and 250 pM for ET-3, whereas particulate membranes had weaker affinities (Ki = 410 pM for ET-1, 2500 pM for ET-3). Calcium channel blockers such as nicardipine, verapamil and diltiazem did not affect the binding of 125I-ET-1. Affinity labeling of the particulate and solubilized membranes with CHAPS revealed a specific binding protein with a Mr of 32,000.     

Solubilization and characterization of active somatostatin receptors from rat pancreas

Somatostatin receptors were solubilized from rat pancreatic membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). The binding of an iodinated somatostatin analog [125I-Tyr3]SMS to the soluble fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of somatostatin binding sites with a Kd of 0.3 nM and a Bmax of 210 fmol/mg. As observed with membrane-bound receptors, soluble binding receptors were sensitive to the GTP analog GTP gamma S indicating that they are functionally linked to a G protein. A molecular weight of about 400,000 was determined for soluble receptors under native conditions by gel filtration. In denaturing gel electrophoresis, photoaffinity labeling of soluble receptors identified a major protein of Mr = 100,000 and two minor proteins of Mr = 56,000 and 21,000. Isoelectric focusing of soluble receptors revealed that the somatostatin receptor is an acidic protein with pI 4.8. The soluble somatostatin receptor is a glycoprotein which can be specifically bound to the wheat germ agglutinin lectin and eluted by triacetyl-chitotriose.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

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.     

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.     

Binding and internalization of atrial natriuretic factor by high-affinity receptors in A10 smooth muscle cells

A10 smooth muscle cells, derived from embryonic rat thoracic aorta, responded to the atrial natriuretic factor (ANF) with increased levels of cyclic GMP. These cells possess high-affinity (apparent Kd = 50 pM) plasma membrane receptors for ANF. Internalization of ANF at 37 degrees C was indicated by the following: approximately 25% of the 125I-ANF associated with the cells at elevated temperatures could not be dissociated from the surface of the cells, but could be released by permeabilization with saponin, and the amount of nondissociable ANF increased in the presence of chloroquine. In whole cells and in membranes, a single polypeptide of 60,000 Da was specifically labeled by a photoaffinity analog of 125I-ANF, as well as by crosslinking, and an IC50 of 80 pM for inhibition of the labeling by ANF was observed. The ANF receptor in A10 cells was distinguished from that in rabbit aorta by its high affinity for shorter and linear analogs of ANF, as well as by a different photolabeling pattern.     

Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells

The biochemical characteristics of specific receptor molecules for diphtheria toxin on the surface of two toxin-sensitive cell lines (Vero and BS-C-1) were examined. Diphtheria toxin was found to bind to a number of different proteins in Nonidet P-40 solubilized extracts of 125I-labeled cells. In contrast, permitting diphtheria toxin to bind first to labeled intact cells, which were subsequently solubilized and subjected to immunoprecipitation with anti-diphtheria toxin, resulted in a far more restricted profile of diphtheria toxin-binding proteins that possessed Mrs in the range of 10,000-20,000. Direct chemical cross-linking of radioiodinated diphtheria toxin to cell surface proteins resulted in the appearance of several predominant bands possessing Mrs of approximately 80,000. The Mr approximately 80,000 complexes were shown to be composed of radiolabeled diphtheria toxin (Mr 60,000) and unlabeled Mr approximately 20,000 cellular proteins. These complexes were judged to be a result of specific binding in that their appearance could be preferentially inhibited by the addition of a 100-fold excess of unlabeled diphtheria toxin. The formation of the Mr approximately 80,000 complexes was sensitive to prior trypsin treatment of the cells and to known inhibitors of diphtheria toxin binding. Furthermore, prior incubation of the cells with diphtheria toxin at 37 degrees C ("down regulation") markedly and specifically reduced the subsequent formation of the Mr approximately 80,000 cross-linked complexes, and these down-regulated cells were less sensitive to diphtheria toxin in cytotoxicity assays. Further incubation of down-regulated cells at 37 degrees C restored their ability to form Mr approximately 80,000 complexes; this regeneration requires protein synthesis and restores the cells' sensitivity to diphtheria toxin-mediated cytotoxicity. These results strongly suggest that a Mr 10,000-20,000 cell surface protein is, or constitutes a portion of, the functional diphtheria toxin receptor.     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.