Monoiodo-[Trp11]neurotensin, a highly radioactive ligand of neurotensin receptors. Preparation, biological activity, and binding properties to rat brain synaptic membranes

Iodination of [Trp11]neurotensin, a neurotensin analogue in which tyrosine 11 has been substituted by a tryptophan, led to the incorporation of one or two iodine atoms on the single tyrosine residue in position 3. Both mono- and diiodinated derivatives were purified by ion exchange chromatography and their biological activity in an in vitro bioassay involving rat ileum was found to be similar to that of native neurotensin. The 125I-labeled monoiodo derivative of [Trp11]neurotensin bound specifically and reversibly to rat brain synaptic membranes. The binding isotherm was biphasic and could be described by postulating the existence of two different classes of independent binding sites with dissociation constants of 0.1 and 4.7 nM. The specificity of a series of neurotensin analogues for both high and low affinity binding sites was the same as that previously observed in other neurotensin radioreceptor assays. The low affinity binding sites appeared to be similar to the single class of sites described in other binding studies. The high affinity binding sites which were not previously detected might represent either a new class of neurotensin receptors or a high affinity state for a fraction of a single population of neurotensin receptors.     

Covalent labeling of neurotensin receptors in rat gastric fundus plasma membranes

Neurotensin receptors from plasma membranes of rat gastric fundus smooth muscle were specifically and covalently labeled either by using the photoreactive analogue ¹²⁵I-labeled azidobenzoyl (Trp¹¹)-neurotensin or by cross-linking (monoiodo-Tyr³)neurotensin to the membrane preparation by means of disuccinimidyl suberate. Analysis of plasma membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the same protein band with an apparent molecular weight of 110,000 was specifically labeled by both methods. This band consisted of a single chain protein since its apparent size was found to be the same with or without reduction of membrane samples before electrophoresis. Only neurotensin and its biologically active analogues were able to protect plasma membranes against specific labeling of the protein band of molecular weight 110,000. Comparison of these results with those obtained from rat brain synaptic membranes shows that although rat central and peripheral neurotensin receptors exhibit similar specificities towards a series of neurotensin analogues, their subunit structures are different.     

Identification of the glucagon receptor by covalent labeling with a radiolabeled photoreactive glucagon analogue

The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.     

Solubilization and characterization of active neurotensin receptors from mouse brain

Neurotensin receptors were solubilized from mouse brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The binding of 125I-labeled [Tyr3]neurotensin to the soluble fraction was time-dependent, saturable, and reversible. Unlabeled neurotensin and its analogues acetylneurotensin (8-13), neurotensin (9-13), and neurotensin (1-12) competitively antagonized the binding of 125I-labeled [Tyr3]neurotensin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane-bound receptors. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of neurotensin binding sites with a Kd of 0.36 nM and a Bm of 63 fmol/mg. As already observed with membrane-bound receptors, the affinity of neurotensin for the soluble binding activity was decreased by Na+ ions. By contrast, soluble receptors were no longer sensitive to GTP and the antihistamine drug levocabastine. A molecular weight of about 100,000 was determined for soluble neurotensin receptors both under native conditions by gel filtration on Ultrogel AcA 34 and under denaturating conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling.     

Purification of the neurotensin receptor from mouse brain by affinity chromatography

The neurotensin receptor was purified from newborn mouse brain by affinity chromatography. Active neurotensin binding sites were solubilized from brain homogenates using the nondenaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in the presence of cholesteryl hemisuccinate. Chromatography of the soluble extract on SP-Sephadex C-25 and hydroxylapatite eliminated 50% of proteins without loss of neurotensin binding activity. This prepurified material was loaded into an affinity column prepared by coupling neurotensin (2-13) to glutaraldehyde-activated Ultrogel AcA22. Nonspecifically adsorbed proteins were eliminated by extensive washing, and the receptor was eluted with a buffer containing 1 M NaCl, 0.1% CHAPS, and 0.02% cholesteryl hemisuccinate. After desalting, the purified receptor bound 125I-labeled neurotensin with a dissociation constant of 0.26 nM and retained its specificity towards a series of neurotensin analogues. The desalted NaCl eluate appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major band of molecular weight 100,000 which was identified as the receptor by affinity labeling with 125I-labeled neurotensin in the presence of disuccinimidyl suberate. The purity of the mouse brain receptor eluted from the affinity column was estimated to be 78%. Electroelution of the 100-kDa protein band gave an homogenous preparation of receptor. Very similar results were obtained with CHAPS-solubilized neurotensin receptors from rat and rabbit brain.     

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

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

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Radiolabeled Ligands Specific for the G Protein-Coupled State of Neurotensin Receptors

Abstract: Radiolabeled analogues of neuromedin N have been prepared by acylation of the α, ε1, and ε2 amino groups of [Lys²]neuromedin N (Lys-Lys-Pro-Tyr-Ile-Leu) either with the ¹²⁵I-labeled Bolton-Hunter reagent or with N -succinimidyl[2,3-³H]propionate. The binding properties of the purified analogues toward newborn mouse brain homogenate or toward membranes of cells transitorily (COS) or permanently (AA1) transfected with the cloned rat brain neurotensin receptor cDNA were evaluated and compared with those of radiolabeled neurotensin. The α-modified analogue of [Lys²]neuromedin N behaves exactly like neurotensin in these binding experiments, whereas the ε1- and ε2-modified analogues selectively recognize the fraction of neurotensin binding sites that is sensitive to GTPγS. The proportion of neurotensin receptors coupled to GTP binding proteins is ∼50% in membranes of newborn mouse brain or of AA1 cells that respond to neurotensin by an increase of the intracellular inositol trisphosphate concentration. By contrast, membranes of transitorily transfected COS cells that do not respond to neurotensin exhibit very low levels of GTP-sensitive receptors labeled with the ε1- or ε2-modified analogues. These radiolabeled peptides offer new tools to selectively detect active neurotensin receptors.     

Identification of a receptor for atrial natriuretic factor in rabbit aorta membranes by affinity cross-linking

Binding sites in rabbit aorta membranes for atrial natriuretic factor (ANF) have been specifically and covalently labeled by two methods. In the first, the photoreactive analog of ANF, 125I-azidobenzoyl-ANF, was synthesized and used to photoaffinity label ANF receptors. In the second, 125I-ANF was covalently attached to its binding site by treatment of the 125I-ANF-receptor complex with bifunctional cross-linking agents. Analysis of the labeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that by both methods the same three protein bands were labeled. These bands had apparent molecular masses of 60,000, 70,000, and 120,000 daltons. With the photoaffinity label, half-maximal inhibition of labeling of each of these bands was achieved when approximately 200 pM of unlabeled ANF was included in the binding assay. These results suggest that these three different polypeptides are specific components of ANF receptors in rabbit aorta membranes.     

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.     

The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage

Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.     

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.     

High-Affinity Neurotensin Binding Sites in Differentiated Neuroblastoma N1E115 Cells

This paper describes the interaction of neurotensin with mouse neuroblastoma N1E115 cells. Neurotensin binding sites are undetectable in nondifferentiated neuroblastoma cells. They appear during cell differentiation in the presence of a low serum concentration and dimethyl sulfoxide, and reach a maximal level after 50-60 h of incubation under these conditions. The binding of monoiodo[Trp11]neurotensin to homogenates of differentiated N1E115 cells is specific, saturable, and reversible. The interaction is characterized by a dissociation constant of 150 pM and a maximal binding capacity of 9 fmol/mg of protein at 0 degrees C, pH 7.5. These binding parameters, as well as the specificity toward a series of neurotensin analogues, are similar for neurotensin receptors in N1E115 cells and for the high-affinity binding sites that had been previously characterized in rat brain synaptic membranes by means of the same radiolabeled ligand. The presence of high-affinity binding sites for neurotensin in the neuroblastoma N1E115 provides a useful model to study the cellular responses that are generated by the association of neurotensin to its receptor in electrically excitable cells.     

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.     

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.     

Saturable binding to cell membranes of the presynaptic neurotoxin, beta-bungarotoxin.

Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

Disulfide bonds within the alpha-subunit of insulin receptors in rat liver and brain membranes

We have characterized inter- and intrasubunit disulfide bonds of insulin receptors using reductant-treated rat liver and brain membranes. In autoradiograms of 125I-insulin cross-linked to both membranes pretreated with dithiothreitol, the intensity of affinity-labeled bands of the alpha beta-heterodimer and alpha-subunit was increased. Interestingly, labeled 120 and 110 kDa bands considered to be the alpha-subunit in partially reduced liver and brain membranes moved to 130 and 120 kDa bands under further reduced conditions, respectively. Double electrophoresis of each partially reduced band in the presence of reductants clearly demonstrates that the alpha-subunit of insulin receptors contains intrasubunit disulfide bonds.     

The effect of reducing agents on the structure of mammalian beta-adrenergic receptors

Under reducing conditions (5% beta-mercaptoethanol) the mammalian beta-adrenergic receptor binding site from both beta 1 (porcine heart membranes) and beta 2 receptors (hamster lung and rat erythrocyte membranes) appears to reside on peptides of Mr 62,000-65,000 as determined by photoaffinity labeling with p-azido-m-[125I]iodobenzylcarazolol and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When similar experiments are performed in these same systems under a variety of non-reducing conditions, there are minimal changes in the apparent molecular weight of both the beta 1- and beta 2-adrenergic receptor binding subunits and no specifically labeled higher molecular weight proteins are observed suggesting that there are no disulfide linked subunits in mammalian beta-adrenergic receptors.