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.     

Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.     

Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide

The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh.     

New specific radioligand for one subpopulation of brain somatostatin receptors

Cyclic octapeptide analogues of somatostatin (SS) like SMS 201-995 [H-(D) Phe-Cys-Phe-(D) Trp-Lys-Thr-Cys-Thr(ol)] or its Tyr3-derivative 204-090, displaced [125I-Tyr11]-SS 100% from pancreatic membranes but only 62-75% from brain membranes; the remaining sites were displaced by SS. These data indicate that some mini-somatostatins bind to a subpopulation of SS receptors in rat brain. The iodinated Tyr3-derivative (125I-204-090) can be considered a selective radioligand for one rat brain SS receptor subpopulation: It shows saturable and high affinity binding (KD = 0.29 nM; Bmax = 350 fmoles/mg protein) to rat cortex. The pharmacological properties of 125I-204-090 binding sites are similar to those of [125I-Tyr11]-SS sites. Distribution of these sites correspond to SS receptor-rich areas such as cortex, hippocampus, striatum, pituitary, pancreatic beta-cell. SS as well as SMS 201-995 bind to these sites with high affinity. The stability and high specific vs non-specific binding ratio makes 204-090 a radioligand of choice to measure this SS receptor subpopulation in CNS but also the SS receptors in pituitary and pancreas.     

The processing of receptor-bound [125I-Tyr11]somatostatin by RINm5F insulinoma cells

The peptide somatostatin (SRIF) is secreted by delta cells of the endocrine pancreas and inhibits the secretion of insulin from pancreatic beta cells. We have previously shown that [125I-Tyr11]SRIF binds to specific, high affinity receptors on RINm5F insulinoma cells and that these receptors mediate the action of SRIF to inhibit insulin release. In the present study we investigated the processing of receptor-bound [125I-Tyr11]SRIF in this clonal cell line. Surface-bound and internalized peptides were distinguished by the ability of an acid/salt solution (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) to dissociate only exposed ligand-receptor complexes. Surprisingly, greater than 80% of saturably bound [125I-Tyr11]SRIF was removed by this acid wash independent of the time or temperature of the binding incubation. In contrast, the processing of receptor-bound [125I]EGF (epidermal growth factor) in RINm5F cells was markedly temperature-dependent. Although over 90% of saturably bound [125I]EGF was dissociated by acid after a 4 degrees C binding incubation, less than 10% was removed by acid treatment after 37 degrees C binding. The radioactivity released upon dissociation of receptor-bound [125I-Tyr11]SRIF was analyzed by high performance liquid chromatography and shown to consist of a mixture of intact peptide (40%) and [125I]tyrosine (60%). However, neither the rate of [125I-Tyr11]SRIF dissociation nor its degradation were affected by NH4Cl, methylamine, or leupeptin at concentrations which inhibited the lysosomal degradation of [125I] EGF. Of 11 other protease inhibitors tested, only the metalloendoprotease inhibitor, phosphoramidon, substantially reduced the degradation of receptor-bound [125I-Tyr11]SRIF. These data indicate that, unlike [125I] EGF, receptor-bound [125I-Tyr11]SRIF is not rapidly internalized by RINm5F cells and is degraded by a nonlysosomal process which may involve a metalloendoprotease.     

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes.

The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.     

Characterization of kinin binding sites: identity of B2 receptors in the epithelium and the smooth muscle of the guinea pig ileum.

Binding of [125I-Tyr8]bradykinin (BK) was measured in homogenates of epithelial and smooth muscle layers of the guinea pig ileum. Binding assays were performed at 4 degrees C for 40 min (smooth muscle) or 90 min (epithelium) in 25 mM PIPES buffer at pH 6.8 in the presence of 1 mM 1,10-phenanthroline, 140 micrograms/mL bacitracin, 1 mM captopril, 1 mM dithiothreitol, and 0.1% bovine serum albumin. Specific binding of [125I-Tyr8]BK (0.32 nM) to epithelial and smooth muscle cell membranes was linearly related to protein concentration between 0.05 and 0.5 mg/mL. Equilibrium experiments showed that specific binding of [125I-Tyr8]BK was saturable and Scatchard analysis indicated the presence of a high affinity site with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg of protein in the epithelial cell membranes. In smooth muscle membranes, Kd was 1.8 nM and the maximum number of binding sites was 58 fmol/mg of protein. Unlabelled peptides, namely bradykinin, [Tyr8]BK, [Hyp3]BK, D-Arg[Hyp3]BK, [Hyp3,Tyr(Me8)]BK, and kallidin displaced [125I-Tyr8]BK binding while other peptides, angiotensin II and substance P, had no effect. A series of B2-receptor antagonists displaced [125I-Tyr8]BK from specific binding sites with IC50 values ranging from 16 to 152 nM on epithelial cell membranes; similar values were obtained from smooth muscle cell membranes. These findings suggest that the binding sites in both preparations are of the B2 type. B1-receptor agonists and antagonists were found to be inactive at concentrations up to 10(-4) M. Results obtained in the two preparations were compared and a positive highly significant correlation was demonstrated between the two sets of data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with tetrafluorophenyl azide-maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites.

Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha subunits) and with the delta subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta subunit to both alpha and beta subunits. Reaction of the mutants with TFPAM's also cross-linked delta to alpha and beta and in addition formed covalent links between Cys10 of the epsilon subunit and the gamma subunit and between Cys108 of the epsilon subunit and the alpha subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta cross-linked products. In the presence of ATP+EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary somatostatin receptors. Characterization by binding with a nondegradable peptide analogue

Somatostatin receptors in the rat pituitary gland were characterized by binding analysis with a radioiodinated high affinity somatostatin analogue, 125I-Tyr1[D-Trp8]somatostatin. Receptor binding of this derivative reached equilibrium at 30 min and was maintained at a plateau for at least 60 min. Two L-Trp8- labeled somatostatin analogues. 125I-Tyr1- and [125I-Tyr11]somatostatin, displayed less stable and lower specific uptake and higher nonspecific binding. In contrast to the rapid degradation of the L-Trp8 ligands during binding assay, 125I-Tyr1]D-Trp8]somatostatin retained more than 80% of its binding activity after 90 min of incubation with pituitary particles. Pituitary particles bound 125I-Tyr1]D-Tyr8]somatostatin with high affinity (Ka = 8.6 +/- 1.2 X 10(9) M-1) and capacity of 54.4 +/- 2.6 fmol/mg. These binding sites showed specificity for the native peptide and its active analogues, and other peptide hormones, including angiotensin II, thyrotropin-releasing hormone, vasopressin, oxytocin, substance P, and gonadotropin-releasing hormone, did not inhibit tracer binding. A good correlation was observed between the binding affinities of several somatostatin analogues and their potencies as inhibitors of growth hormone release in rat pituitary cells. These findings emphasize the physiological importance of the pituitary somatostatin receptor in mediating the inhibitory action of the peptide on growth hormone release. The use of Tyr1[d-Trp8]somatostatin as a labeled ligand permits accurate determinations of the binding affinity and concentration of receptors for somatostatin in the normal pituitary gland and provides a basis for further studies of somatostatin receptor regulation and receptor-mediated cellular effects of the tetradecapeptide.     

Identifying the lipid-protein interface of the alpha4beta2 neuronal nicotinic acetylcholine receptor: hydrophobic photolabeling studies with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.     

The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.     

Chemical cross-linking of somatostatin receptors in rat adrenal cortex

Adrenocortical somatostatin receptors have been shown to interact with somatostatin-14 (S-14) and somatostatin-28 (S-28). To determine whether these peptides interact with the same or different receptor proteins, we chemically cross-linked these receptors using disuccinimidyl suberate to radioligands prepared from tyrosinated S-14 and S-28 analogs. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and subsequent autoradiography of [125I-Tyr11] S-14 and [Leu8, D-Trp22, 125I-Tyr25] S-28 cross-linked to their binding sites following solubilization in the presence of 50 mM DTT revealed the presence of a single labelled protein of Mr = 200,000. When the cross-linked material was treated under non-reducing conditions, this band was not observed. Furthermore, addition of excess S-14 and S-28 at the time of binding inhibited the incorporation of both radioligands into the receptor protein. These results demonstrate that adrenocortical membrane receptors for somatostatin contain a single receptor protein sub-unit or sub-units of identical size which interact with both S-14 and S-28.     

Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes.

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.     

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.     

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.     

The C-terminal domain of Escherichia coli ribosomal protein L7/L12 can occupy a location near the factor-binding domain of the 50S subunit as shown by cross-linking with N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide.

All large ribosomal subunits contain two dimers composed of small acidic proteins that are involved in binding elongation factors during protein synthesis. The ribosomal location of the C-terminal globular domain of the Escherichia coli ribosomal acidic protein L7/L12 has been determined by protein cross-linking with a new heterobifunctional, reversible, photoactivatable reagent, N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propionamide . Properties of this reagent are described. It was first radiolabeled with 125I and then attached through the formation of a disulfide bond to a unique cysteine of L7/L12, introduced by site-directed mutagenesis at residue 89. Intact 50S ribosomal subunits were reconstituted from L7/L12-depleted cores and the radiolabeled L7/L12Cys89. Irradiation of the reconstituted subunits resulted in photo-cross-linking between residue 89 and other ribosomal components. Reductive cleavage of the disulfide cross-link resulted in transfer of the 125I label from L7/L12Cys89 to the other cross-linked components. Two radiolabeled proteins were identified, L11 and L10. The location of both of these proteins is well established to be at the base of the L7/L12 stalk near the binding sites for the N-terminal domain of both L7/L12 dimers, and for elongation factors. The result indicates that L7/L12 can have a bent conformation bringing the C-terminal domain of at least one of the L7/L12 dimers at or near the factor-binding domain. The cross-linking method with radiolabeled N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide should be applicable for studies of other multicomponent complexes that can be reconstituted.     

Binding of monoiodinated neuropeptide Y to hippocampal membranes and human neuroblastoma cell lines

Monoiodinated radioligands of the homologous 36-amino acid peptides, neuropeptide Y (NPY) and peptide YY, were prepared by reverse phase high performance liquid chromatography with isocratic elution. [125I-Tyr1]- and [125I-Tyr36]monoiodoNPY bound equally well to a single class of high affinity binding sites on synaptosomal membranes prepared from porcine hippocampus (Kd = 1.0 X 10(-10) M) whereas iodine substitution in Tyr27, for example, partly interfered with the receptor binding. The receptors on the hippocampal membranes did not distinguish between neuropeptide Y and peptide YY either in their monoiodinated or in their unlabeled forms. Six out of twelve human neuroblastoma cell lines had high affinity binding sites for monoiodinated NPY ranging from 2 to 145 X 10(3) sites per cell. The NPY binding to three of the cell lines, SMS-MSN, SMS-KAN, and CHP-234 was of relatively high affinity (Kd = 1.3 to 6.1 X 10(-10) M), and, as in the hippocampal membranes, the long C-terminal fragment, NPY(13-36)peptide was also a relatively potent ligand for these receptors. Two other neuroblastoma cell lines, MC-IXC and CHP-212, expressed NPY receptors characterized by a lower affinity (Kd = 4.8 and 24.6 X 10(-9) M) and negligible cross-reactivity with the C-terminal fragment. It is concluded that monoiodinated radioligands of the tyrosine-rich neuropeptide Y can be prepared and that receptors for these ligands in two apparently different subtypes are found on a series of human neuroblastoma cell lines.     

Mastoparan interacts with the carboxyl terminus of the alpha subunit of Gi

Mastoparan, a peptide toxin from wasp venom, stimulates guanine nucleotide binding and hydrolysis by G proteins. To elucidate the site of mastoparan-G protein interaction, we utilized a polyclonal antibody (R16,17) directed against the carboxyl terminus of the Gi alpha subunit to develop a competitive enzyme-linked immunosorbent assay. We investigated the ability of mastoparan to influence R16,17 antibody binding to G protein alpha subunits in a purified preparation of brain Gi and in neutrophil membrane extracts. Mastoparan antagonized the ability of R16,17 to detect G protein alpha subunits with an IC50 of 15 microM in the purified preparation and with an IC50 of 1 microM for the predominant G protein population in membrane extracts. This reduction was not seen when an unrelated peptide or a peptide of similar charge composition to mastoparan was used in place of mastoparan in the assay. Additionally, antibody R16,17 blocked up to 85% of mastoparan-stimulated GTPase activity. Taken together, these data indicate that the interaction of mastoparan with G protein depends in part on the carboxyl terminus of Gi alpha. Pertussis toxin-catalyzed ADP-ribosylation of Gi alpha markedly inhibited mastoparan-stimulated GTPase activity but only slightly attenuated the ability of mastoparan to recognize G protein. These data suggest that ribosylation inhibits mastoparan-induced G protein activation by a mechanism distinct from the ability of mastoparan to physically interact with G protein. Since mastoparan is thought to mimic hormone-liganded receptors, these findings may be applicable to the mechanism of receptor-Gi protein uncoupling that results from ADP-ribosylation of the G protein.