High and low affinity receptors mediate growth effects of gastrin and gastrin-Gly on DLD-1 human colonic carcinoma cells

Gastrin (G17) and N-carboxymethylgastrin (G17-Gly) have been shown to stimulate the growth of colon cancer cells both in vivo and in vitro. The identity of the receptor mediating these effects is controversial. A recent study demonstrated the presence of a low affinity binding site for G17 and G17-Gly on the DLD-1 human colon cancer cell line. The goal of the current study was to further investigate the role of this receptor in mediating the growth-promoting effects of gastrin peptides. Binding of [Leu(15)]G17 and [Leu(15)]G17-Gly to DLD-1 cell membranes in competition with [(3)H]G17-Gly was examined. Binding of [(3)H]cholecystokinin-8 (CCK8) to DLD-1 cell membranes was also assessed. Whole cell binding experiments were carried out using [(125)I-Tyr(12),Leu(15)]G17-Gly. In addition, the ability of [Leu(15)]G17 and [Leu(15)]G17-Gly to stimulate cell growth, as determined by cell counting, was tested. [Leu(15)]G17 and [Leu(15)]G17-Gly competed with [(3)H]G17-Gly at both a high and a low affinity site on DLD-1 membranes. The IC(50) values for [Leu(15)]G17 were 6.0 x 10(-8) M and 6.9 x 10(-6) M while those for [Leu(15)]G17-Gly were 3.2 x 10(-9) M and 4.9 x 10(-6) M. [(3)H]CCK8 did not bind to either site. [Leu(15)]G17-Gly also competed with [(125)I-Tyr(12),Leu(15)]G17-Gly at both a high and a low affinity site on DLD-1 cells with similar affinities as observed with membranes. [Leu(15)]G17 and [Leu(15)]G17-Gly significantly stimulated the growth of DLD-1 cells in a dose-dependent and biphasic manner. The binding profiles of the peptides tested suggest that these sites are different from previously identified wild-type and mutant CCK(1) or CCK(2) receptors.     

Gastrin 1-6 promotes growth of colon cancer cells through non-CCK receptors

Our previous studies have shown that stimulation of proliferation of DLD-1 and HT29 human colonic cancer cells by nanomolar gastrin (G17) and carboxymethyl gastrin (G17Gly) and reversal of growth by micromolar G17 and G17Gly involves binding sites which can neither be CCK1 nor CCK2 receptors; the N terminal fragment, G17(1-12), is sufficient to increase the number of HT-29 cells by binding the higher affinity binding site but is without a suppressing effect through the lower affinity site. In this study with DLD-1 cells, competitive binding using 125I-G17(1-12) showed that G17(1-12) binds both high and low affinity sites, as do G17 and G17Gly. G17(1-6)-NH2, even without the central-to-C-terminal portion of G17, was still able to bind a single site and to promote a dose-dependent increase in cell number at nanomolar concentrations. The results indicate the presence of a non-CCK receptor on human colonic cancer cells which could mediate the tumor-promoting activity of the N-terminal-to-central portion of G17Gly which, unlike G17, is produced by such cells.     

Importance of the C-terminal phenylalanine of gastrin for binding to the human CCK(2) receptor.

The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15,Ala17]G(11-17), [Leu15,Abu17]G(11-17), [Leu15,Val17]G(11-17), [Leu15,Leu17]G(11-17), [Leu15,Cha17]G(11-17), [Leu15,Trp17]G(11-17), [Leu15,Tic17]G(11-17), [Leu15, d-Phe17]G(11-17) and [Leu15,p-X-Phe17]G(11-17), where X = F, Cl, Br, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g+ or g- conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.     

Probing peptide backbone function in bombesin. A reduced peptide bond analogue with potent and specific receptor antagonist activity

Each peptide bond CONH group in the most important COOH-terminal octapeptide region of [Leu14]bombesin was replaced by a CH2NH group using recently developed rapid solid-phase methods. The resulting analogues were then examined for amylase releasing activity in guinea pig pancreatic acini and for their ability to inhibit binding of [125I-Tyr4]bombesin to acinar cells. Replacement of the Trp8-Ala9, Gly11-His12, and His12-Leu13 peptide bonds resulted in about 1000-, 200-, and 300-fold losses in both amylase releasing activity and binding affinity. The Val10-Gly11 replacement, however, retained 30% potency relative to the parent peptide. Ala9-Val10 and Leu13-Leu14 bond replacement analogues exhibited no detectable amylase releasing activity but were still able to bind to acini with Kd values of 1060 and 60 nM, respectively (compared to 15 nM for [Leu14]bombesin itself). Subsequently, both analogues were demonstrated to be competitive inhibitors of bombesin-stimulated amylase release with IC50 values of 937 and 35 nM, respectively. [Leu14-psi-CH2NH-Leu13]Bombesin exhibits a 100-fold improvement in binding affinity compared to previously reported bombesin receptor antagonists and showed no affinity for substance P receptors. It was also a potent inhibitor of bombesin-stimulated growth of murine Swiss 3T3 cells with an IC50 of 18 nM. In terms of a bombesin receptor-binding conformation, these results may aid in the delineation of intramolecular hydrogen-bonding points and the eventual design of improved, conformationally restricted analogues.     

Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain

Metorphamide is a [Met]-enkephalin-containing opioid octapeptide with a C-terminal alpha-amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for mu opioid (morphine) receptors, a somewhat lesser affinity for kappa opioid receptors, and a relatively low affinity for delta opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared with the concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]-enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6-Gly7-Leu8 from proenkephalin; alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), dynorphin A(1-17), and dynorphin B from prodynorphin; and [Leu]-enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalin-derived opioid peptides. They were, however, similar to the concentrations of the prodynorphin-derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin-derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's mu opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous mu opioid receptor ligand.     

Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5)

We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 +/- 0.04 nM for BIBO3304 vs. 2.4 +/- 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 microM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor.     

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPARγ is expressed at considerable levels in human colon cancer cells. This suggests that PPARγ expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPARγ expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPARγ mRNA and protein in these cells were in the order HT-29>LOVO>Caco-2>DLD-1. We also found that PPARγ overexpression promoted cell growth inhibition in PPARγ lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPARγ expression and the cells' sensitivity for proliferation.     

The roles of tyrosines 24, 31, and 60 in the high affinity binding of insulin-like growth factor-I to the type 1 insulin-like growth factor receptor

A series of insulin-like growth factor I (IGF-I) structural analogs in which one or more of the three tyrosine residues were replaced with nonaromatic residues were produced and their binding properties characterized. The single point mutations, [Leu24]IGF-I, [Ala31]IGF-I, and [Leu60]IGF-I result in an 18-, 6-, or 20-fold loss in affinity, respectively, for the type 1 IGF receptor. Multiple mutations, [Ala31,Leu60]IGF-I, [Leu24, Ala31]IGF-I, [Leu24, Leu60]IGF-I, or [Leu24, Ala31, Leu60]IGF-I result in a 520-, 240-, 1200-, or greater than 1200-fold loss in affinity, respectively, at the type 1 IGF receptor. In contrast, none of the analogs display greater than a 2-fold loss in affinity for the acid-stable human serum binding proteins. At the insulin receptor, [Ala31]IGF-I and [Leu24]IGF-I are equipotent to and 5-fold less potent than IGF-I, whereas [Leu60]IGF-I and the multiple mutation analogs are inactive up to 10 microM. Analogs [Leu24]IGF-I, [Ala31]IGF-I, and [Leu24, Ala31]IGF-I are equipotent to IGF-I at the type 2 IGF receptor, whereas all analogs containing Leu60 demonstrate little measurable affinity at this receptor. Thus, Tyr24, Tyr31, and Tyr60 are involved in the high affinity binding of IGF-I to the type 1 IGF receptor, while Tyr60 is important for maintaining binding to the type 2 IGF receptor.     

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.     

Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62-67.

Five structural analogs of human insulin-like growth factor II (IGF II), [Leu27]IGF II, [Glu27]IGF II, des(62-67)IGF II, des(62-67)[Leu27]IGF II and des(62-67)[Glu27]IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E. coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC. These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. [Leu27]IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor. The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

Leu31-Pro34] neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain.

Subtypes of the neuropeptide Y (NPY) receptor in the rat brain were identified by the use of the selective Y-1 analog, [Leu34-Pro34] NPY. In rat brain homogenate binding studies, [Leu31-Pro34] NPY was found to produce a partial inhibition of 100 pM 125I-labeled peptide YY (PYY) binding with a plateau at 50-1000 nM [Leu31-Pro34] NPY resulting in a 70% inhibition of binding. The C-terminal fragment NPY 13-36, a putative Y-2 agonist, exhibited very little selectivity in rat brain homogenates. Scatchard analysis of 125I-labeled PYY binding to rat brain homogenate yielded biphasic plots with Kd values of 40 and 610 pM. Inclusion of 100 nM [Leu31-Pro34] NPY was found to eliminate the low affinity component of 125I-labeled PYY binding leaving a single, high affinity binding site with a Kd of 68 pM. In autoradiographic studies, displacement curves indicated that [Leu31-Pro34] NPY completely inhibited binding in the cerebral cortex with little effect on the binding in the hypothalamus. On the other hand NPY 13-36 inhibited binding in the hypothalamus at low concentrations but required higher concentrations to inhibit binding in the cerebral cortex. Other brain regions such as the hippocampus, appeared to contain both subtypes. Subsequent to these studies, a quantitative autoradiographic map was conducted using 50-100 pM 125I-labeled PYY in the presence and absence of [Leu31-Pro34] NPY which produced a selective displacement of binding in certain distinct brain regions. These areas included the cerebral cortex, certain thalamic nuclei and brainstem while ligand binding was retained in other brain regions including the zona lateralis of the substantia nigra, lateral septum, nucleus of the solitary tract and the hippocampus. Numerous brain regions appeared to contain both receptor subtypes. Therefore, the Y-1 and Y-2 receptor subtypes exhibited a somewhat distinct distribution in the brain. In addition, 125I-labeled PYY appears to label the Y-2 receptor with relatively higher affinity when compared to the Y-1 receptor.     

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.     

Glycine-extended gastrin promotes the invasiveness of human colon cancer cells.

Colorectal cancers express significant amounts of immature glycine-extended gastrin (G-Gly) and G-Gly is able to stimulate cell proliferation in colonic cell lines and mucosa. Here we wished to investigate whether G17-Gly promote the invasiveness of LoVo human colonic cancer cells, a process which requires degradation of extracellular matrix by proteases and concomitant induction of cell migration. We confirmed that LoVo cells express gastrin and gastrin/CCK-B receptor mRNAs. We showed that these cells secrete matrix metalloproteinase (MMP)-1, -2, and -9. The function of MMP being to degrade components of extracellular matrix, they may thus favor cell migration. As compared to controls, G17-Gly (10(-7) to 10(-12) M) significantly enhanced about two to three times the LoVo cell migration through Matrigel, an artificial basement matrix barrier. Moreover, G17-Gly increased and gastrin/CCK-B receptor antagonists decreased MMP secretion in conditioned culture media of LoVo cells. Our findings show that physiological doses of incompletely processed form of gastrin induce the invasiveness of tumor cells in vitro and suggest a novel potential role for this peptide in the metastatic process of colonic cancers in vivo.     

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.     

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Increased amidation

We have used antisera directed towards eight different portions of the proenkephalin molecule to examine the processing rates and patterns of proenkephalin-derived peptides in chromaffin cell cultures in the presence and absence of reserpine. Reserpine treatment produced profound effects on the molecular weight profile of nearly all enkephalin-containing peptides. Increased production of low molecular weight immunoreactive [Met5]enkephalin, [Leu5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, and [Met5]enkephalin-Arg6-Phe7 was observed in reserpine-treated cultures; immunoreactivity corresponding to several intermediate sized enkephalin-containing peptides such as Peptide B and the high molecular weight [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive peptide was decreased. The production of two amidated opioid peptides, amidorphin and metorphamide, was greatly accelerated in the presence of reserpine. The increased levels of low molecular weight enkephalins could not be accounted for by assuming decreased basal release. These results indicate that reserpine treatment is able to increase the extent of post-translational processing of proenkephalin within chromaffin cells.     

Receptor selectivity of enkephalin analogs carrying artificial address peptides

The synthetic [Leu]enkephalin analogs YGGFLGP(KS'S'S')2-OMe (S' represents Sar residue, ENK-S'8), YGGFLGP(KPPP)2-OMe (ENK-P8), and YGGFLGP(KA'LA')2-OMe (A' represents 2-aminoisobutyric acid (Aib), ENK-A'8) were designed to investigate the role of membrane affinity in opioid receptor binding. CD measurement in ethanol revealed that ENK-S'8 took a random coil conformation, while ENK-P8 and ENK-A'8 partly adopted a 3(1)- and alpha-helical conformation, respectively. ENK-A'8 was shown to be distributed to lipid bilayer membrane due to the amphiphilic alpha-helical structure, while ENK-S'8 and ENK-P8 stayed in aqueous phase. Opioid receptor affinities ENK-S'8 and ENK-P8 were similar to [Leu]enkephalinamide, indicating that the connection of hydrophilic peptide segments to the C-terminal of [Leu]enkephalinamide did not affect the receptor affinity. On the other hand, delta- and mu-receptor affinities of ENK-A'8 were about 1/20 and 1/6 times, respectively, those of [Leu]enkephalinamide. Therefore, ENK-A'8 has higher selectivity for mu-receptor than [Leu]enkephalinamide. These results are explained in terms of the membrane compartment concept proposed by Schwyzer.     

The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells

Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 cells.