Carboxyl-terminal Peptide Research Articles

Immunotolerance induced by intratesticular antigen priming: expression of TGF-β, Fas and Fas ligand

The authors have previously reported that an injection of Santigen (S-Ag) into rat testes prior to immunization induces systemic tolerance (designated orchidic tolerance) and protects the animals from experimental autoimmune uveoretinitis (EAU) and that the signal for orchidic tolerance induction emigrates from the testis within a few hours after antigen priming of the testis. In order to understand the mechanism by which the signal or signal carrier is generated, they determined in this study changes in immunoreactivity for transforming growth factor-β (TGF-β), IFN-β, IL-2, Fas and Fas ligand in the testis following an injection of S-Ag. Immunoreactivity for TGF-β increased with time, reaching a maximum in six hours and declining thereafter. The time required for the maximum expression of TGF-β coincided well with the time-dependent profile of orchidic tolerance signal generation within the testis. Little or no immunoreactivity was observed for IFN-γ and IL-2 in normal (control) and S-Ag-injected testes. Immunoreactivity for Fas and Fas ligand was detected both in control and experimental testes and did not change appreciably with time following Ag-priming of the testis. Fas immunoreactivity was found in spermatids and virtually absent in the interstitial tissue, while Fas ligand immunoreactivity was primarily associated with the interstitial cells such as Leydig cells. Fas ligand immunoreactivity was very weak, if any, in the germ cells and Sertoli cells. These results suggest that TGF-β and Fas ligand expressed in MHC-positive interstitial cells may play an important role in the generation of orchidic tolerance induction signal. A preliminary study showed that splenocytes preincubated with testis extracts and S-Ag, when transferred to naive rats, induced systemic tolerance in recipient animals. Inclusion of anti-TGF-β or a carboxyl terminal peptide of Fas in the testis extract reduced the potency of incubated splenocytes to induce systemic tolerance in recipient rats. These results indicate that generation of the orchidic tolerance signal does not require the anatomical structure of the testis but is mediated by molecular entities such as TGF-β and Fas ligand.

Structure of recombinant human serum albumin from Pichia pastoris

The structure of recombinant human serum albumin derived from Pichia pastoris (rHSA) was analyzed in detail. Complete amino acid analysis was performed by the phenyl isothiocyanate precolumn labeling method. The amino terminal sequence was determined by the Edman degradation. The carboxyl terminal amino acid was determined by digestion with carboxypeptidase, and the carboxyl terminal peptide fragment was analyzed by electrospray mass spectrometry. The peptide fragments of rHSA digested with Lysylendopeptidase, Endoproteinase Glu-C, or Endoproteinase Asp-N were analyzed by electrospray mass spectrometry. The complete amino acid composition, the terminal sequences and the complete amino acid sequence of rHSA agreed with the primary structure deduced from its cDNA. The elution pattern of reduced and carboxymethylated rHSA digested with Lysylendopeptidase and the elution pattern of intact rHSA digested with pepsin were respectively similar to those of plasma-derived human serum albumin (pHSA). The pattern of CD spectrum of rHSA was identical in both shape and magnitude to that of pHSA. 1H-NMR spectra of rHSA and pHSA in deuterium oxide showed the same signal patterns in the observed region (delta 10.5-0.5 ppm). Cross peaks assigned to the alpha proton-beta proton of Asp-1 (delta 4.2/2.8 ppm) and the delta proton-epsilon proton of lysine residues (delta 2.8-3.2/1.4-2.0) showed the same cross peak patterns and chemical shifts in two-dimensional phase sensitive double-quantum filtered 1H-1H correlation spectra of rHSA and pHSA.

Immunogenicity of a fusion protein linking the beta subunit carboxyl terminal peptide (CTP) of human chorionic gonadotropin to the B subunit of Escherichia coli heat-labile enterotoxin (LTB)

Human chorionic gonadotropin (hCG) is currently under investigation as an antigenic target in both anti-cancer and anti-fertility vaccines. Formulations studied to date show promise in clinical trials for both applications yet are expensive to produce and require frequent administration in order to maintain an effective antibody titer. We have engineered a fusion protein consisting of Escherichia coli heat-labile enterotoxin subunit B (LTB) genetically linked at its C terminus via a nine amino acid linker to the 37 amino acid carboxyl terminal peptide (CTP) of the hCG beta chain. This LTB-CTP fusion protein is stably expressed in bacteria and forms pentamers of full-length protein subunits. Purified LTB-CTP protein induces hCG-specific antibodies in mice without additional adjuvants.

The Properties of the Protein Tyrosine Phosphatase PTPMEG

We previously cloned a cDNA encoding a protein tyrosine phosphatase (PTP) containing sequence homology to protein 4.1, designated PTPMEG. Recombinant protein and amino- and carboxyl-terminal peptides were used to obtain polyclonal antibodies against PTPMEG to identify endogenous PTPMEG in A172 cells and to show that the enzyme is primarily localized to the membrane and cytoskeletal fractions of these cells. We prepared recombinant protein in Sf9 and COS-7 cells to further characterize it. The protein was phosphorylated in both cell types on serine and threonine residues. The multiple sites of phosphorylation were all within the intermediate domain of the protein between amino acids 386 and 503. This region also contains two PEST sequences and two proline-rich motifs that may confer binding to Src homology 3 domains. The recombinant protein was cleaved by trypsin and calpain in this region and thereby activated 4-8-fold as assayed using Raytide as substrate. We immunoprecipitated the protein from human platelets with both amino- and carboxyl-terminal antipeptide antibodies to assess the state of the enzyme in these cells. The full-length molecule was found in extracts from unstimulated platelets, whereas extracts from both calcium ionophore- and thrombin-treated platelets contained proteolyzed and activated forms of the enzyme, indicating that proteolysis by calpain is evoked in response to thrombin. Prior incubation of platelets with calpeptin, an inhibitor of calpain, blocked the agonist-induced proteolysis.

Kappa-casein suppresses melanogenesis in cultured pigment cells.

The effects of bovine milk proteins on melanogenesis in B16 cells were examined. Both whey protein isolate and casein exhibited depigmenting properties. Among the major protein components of milk--including beta-lactoglobulin, alpha-lactalbumin, alpha-, beta-, and kappa-casein--only kappa-casein exhibited the depigmenting effect. However, the carboxyl terminal peptide of kappa-casein, glycomacropeptide, did not show this activity. Also, kappa-casein promoted the proliferation of the cells and inhibited the activity of tyrosinase in the cells. These results indicate that kappa-casein acts as a melanogenesis-suppressing modulator.

Neuropeptide Y: localization in the brain and pituitary of the developing frog (Rana esculenta).

The immunohistochemical localization of neuropeptide Y (NPY)-like peptide has been investigated in the peripheral terminal nerve, brain and pituitary of the frog, Rana esculenta, during development. Soon after hatching, a rather simple NPY-immunoreactive (-ir) neuronal system is present, with elements located mainly in the diencephalon. When hind limbs appear and develop, the NPY-neuronal system undergoes considerable elaboration and NPY-ir perikarya appear in several regions of the telencephalon (dorsal, medial, and lateral pallium; medial septum; medioventral telencephalon; anterior preoptic area), diencephalon (ventromedial, central and posterior thalamic nuclei; suprachiasmatic nucleus; infundibulum), mesencephalon (anteroventral mesencephalic tegmentum), and rhombencephalon (central grey; area of the cerebellar and vestibular nuclei). The frequency of NPY-ir neurons increases during larval development, and then decreases in the anterior preoptic area during the metamorphic climax. Dense plexuses of NPY-ir fibers are formed in several brain areas. NPY-ir fibers are found in the peripheral terminal nerve, and ir-neurons through its course along the ventromedial surface of the olfactory bulbs. NPY-ir fiber projections to the median eminence and pars intermedia derive mainly from the ventral infundibular group of NPY-ir neurons, with a contribution from the suprachiasmatic group of NPY neurons. NPY and carboxyl terminal flanking peptide of proneuropeptide Y coexist in the same neurons throughout the brain. The ontogenetic pattern of NPY-ir neuronal system in the brain of Rana esculenta is remarkably different than that reported for Xenopus laevis.

Transmembrane topology of Escherichia coli H +-ATPase (ATP synthase) subunit a

Escherichia coli H +-ATPase subunit a is a hydrophobic F 0 subunit. To investigate the topology of the subunit in the membrane, we prepared site-specific polyclonal antibodies against amino-terminal (Ser-3 to Leu-16), middle loop (Lys-167 to Gln-181), and carboxyl-terminal (Thr-259 to His-271) peptide segments. Enzyme-linked immunosorbent assay revealed that these antibodies specifically reacted with subunit a of inside-out membrane vesicles, but not with that of right-side-out spheroplasts. Full reactivity appeared when spheroplasts were disrupted with Triton X-100 (0.5%) or by sonication. These results suggest that at least parts of the three peptide segments of subunit a face the cytoplasm. Based on these observations, we propose a novel transmembrane topology of subunit a.

Growth hormone (GH) retardation of muscle damage due to immobilization in old rats. Possible intervention with a new long-acting recombinant GH.

Four weeks immobilization of the right leg of aged rats (26 months old) caused a marked 31% and 27% reduction of muscle mass of the plantaris and soleus muscles, respectively. In animals treated with 0.6 mg/kg body weight of growth hormone (GH), the reduction of weight of the above muscles was only 14.7 and 16.1%, respectively. Biochemical studies of the level of acid phosphatase as a marker of muscle catabolism showed a significant increase of this enzyme in the immobilized muscles. GH treatment had a positive effect in curtailing the increase due to immobilization. Studies on muscle protein oxidation used as another measure of damage in immobilized animals, showed a 400% increase in protein carbonyls in plantaris muscles. GH administration reduced this value significantly. One major issue hampering the clinical use of human GH (hGH) is its short half-life in vivo (14 min). In a previous work it was possible to enhance the in vivo longevity of other hormones such as follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) by fusing carboxyl-terminal peptide (CTP) of the hCG gene to the above hormones. The CTP has four serine-linked oligosaccharides, which have been shown to be important in maintaining the longer half-lives of these hormones. With the above rationale of using the CTP as a general target to increase the potency of bioactive hormones, we have now fused the CTP with hGH. This has provided us with a new successfully constructed recombinant hGH, which is currently being tested for its biological potency and for possible use in aging animals.

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation.

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.

Photolabeling of Prostaglandin Endoperoxide H Synthase-1 with 3-Trifluoro-3-(m-[125I]iodophenyl)diazirine as a Probe of Membrane Association and the Cyclooxygenase Active Site

Previous studies of the crystal structure of the ovine prostaglandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine vesicular gland microsomes with [125I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg277 with trypsin established that [125I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [125I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [125I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [125I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH2 terminus of the protein.

Molecular Cloning and Characterization of a Novel Human Diacylglycerol Kinase ζ

Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGKzeta displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.

Structural analysis of the carboxyl terminal peptide from human chorionic gonadotropin beta-subunit by two-dimensional nuclear magnetic resonance spectroscopy.

Vaccines that target human chorionic gonadotropin (hCG) might be made more effective through greater understanding of the solution three-dimensional structure and behavior of the hormone. A 37-amino acid carboxyl terminal peptide of the hCG beta subunit was synthesized, purified, and analyzed by two-dimensional nuclear magnetic resonance spectroscopy. Double-quantum filtered correlated spectroscopy data on the peptide in water at 4 degrees C reveals 27 multiplets in the peptide fingerprint region, as expected. A nuclear Overhauser effect spectroscopy spectrum shows several intraresidual peaks in the amide region but lacks clearly assignable interresidual signals. By itself in water the carboxyl terminal peptide of hCG lacks defined secondary structure elements and is thus likely a random coil. The presence of beta turns appears possible although neither their existence nor their localization can be confirmed.

The Carboxyl-terminal Domain(111–165) of Vascular Endothelial Growth Factor Is Critical for Its Mitogenic Potency

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.

Preferred sites of glycosylphosphatidylinositol modification in folate receptors and constraints in the primary structure of the hydrophobic portion of the signal.

The divergent carboxyl-terminal signal peptides for glycosylphosphatidylinositol (GPI) membrane anchor attachment in folate receptor (FR) types alpha and beta were characterized. All of the candidate amino acid residues for GPI modification were identified and tested by substituting individually and in combination with amino acids that cannot be modified by GPI. Thus the GPI modification in FR-alpha was decreased to 22% by mutation of Ser234 to Thr but unaltered by changing the other candidate, Gly235, to Met. However, the double mutant FR-alpha Ser234-Thr,Gly235-Met showed half of the GPI modification seen in FR-alpha Ser234-Thr. This result suggests that Ser234 is the preferred GPI modification site, while Gly235 is a minor, alternate GPI modification site. Similarly, in FR-beta, mutation of Asn230 to Gln decreased GPI modification to 32%, while mutation of the other candidate site, Gly237, to Met had no effect. However, mutation at both sites further reduced the GPI modification by a half. A five amino acid carboxyl-terminal deletion (FR-beta delta 5) caused no decrease in the extent of GPI modification. However, the same deletion in FR beta Asn230-Gln decreased the residual GPI modification by 66%. These results suggest that Asn230 is the preferred GPI modification site in FR-beta, while Gly235 offers a minor alternate modification site; consistent with this conclusion is the fact that modification at the downstream site is hindered by its proximity to the carboxyl terminus in FR-beta delta 5. Further, the suggestion that the hydrophobic portion of the GPI signal is a random sequence of neutral amino acids with overall moderate hydrophobicity was tested.(ABSTRACT TRUNCATED AT 250 WORDS)

The Human Urinary Epidermal Growth Factor (EGF) Precursor: ISOLATION OF A BIOLOGICALLY ACTIVE 160-KILODALTON HEPARIN-BINDING PRO-EGF WITH A TRUNCATED CARBOXYL TERMINUS

In this report, we describe the isolation from human urine of a predominant 160-kDa epidermal growth factor (EGF)-immunoreactive glycoprotein that exhibits affinity for heparin. The purification procedure involved concentration and dialysis of 20-30-liter batches of fresh urine on a high capacity ultrafiltration apparatus followed by chromatography on DEAE-Sephacel, heparin-agarose, and Sephacryl S-300. A nearly homogeneous preparation of 160-kDa protein was obtained with a yield of approximately 1 mg of 160-kDa protein from 25 liters of urine. The amino-terminal sequence of the purified 160-kDa protein, H2N-SAPQHXSXPEGTXA-, matched residues 21-34 of the predicted sequence of human prepro-EGF and established that the 160 kDa protein (pro-EGF) is a product of the prepro-EGF gene. Characterization of the carboxyl terminus of the purified protein by digestion with carboxypeptidase B and by immunoblotting with antisera against synthetic carboxyl-terminal and juxtatransmembrane peptides of prepro-EGF indicated that the carboxyl terminus has been truncated at an arginine residue that corresponds, most likely, to the carboxyl-terminal arginine of the EGF moiety. The intact 160-kDa pro-EGF is biologically active as evidenced by its specific binding to the EGF receptor and activation of the EGF receptor tyrosine kinase in A-431 cell membranes. Purified pro-EGF competitively inhibited the binding of 125I-EGF to human fibroblasts, and it stimulated the proliferation of these cells in culture. When immobilized onto culture dishes, the heparin-binding pro-EGF appeared to function both as an adhesion molecule and as a growth factor for serum-free mouse embryo cells.

Distribution of PASII/PMP22 and connexin 32 proteins in the peripheral nervous system

Various mutations of P0, PASII/PMP22 and connexin 32 genes were recently reported in hereditary neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine Sottas disease (DS). However, physiological roles of the proteins in PNS are not well understood. To address the functions of the proteins, we examined their localization in PNS comparatively by immunohistochemical methods. In Western blotting, a polyclonal antibody against the carboxyl terminal peptide of PASII/PMP22 reacted to 20–24 kD bands of PASII/PMP22 in mammalian PNS myelin, but produced no reaction in either mammalian or carp CNS myelin proteins. Monoclonal anti-connexin 32 antibody recognised connexin 32 of a dimer or monomer form in rat and human PNS myelin. By histological examination, PASII/PMP22 expressed dominantly in rat PNS compact myelins, while connexin 32 localized exclusively in the nodes of Ranvier, but not in compact myelins. In cell culture, axonal contact induced a remarkable increase of PASII/PMP22 in the Schwann cell in contrast to faint staining in immature Schwann cells. While localization of connexin 32 is quite different from that of PASII/PMP22, the mutations of the two proteins often induce similar phenotypes of hereditary neuropathies.

Evidence That the Pertussis Toxin-sensitive Trimeric GTP-binding Protein Gi2 Is Required for Agonist- and Store-activated Ca2+ Inflow in Hepatocytes

The role of a trimeric GTP-binding protein (G-protein) in the mechanism of vasopressin-dependent Ca2+ inflow in hepatocytes was investigated using both antibodies against the carboxyl termini of trimeric G-protein alpha subunits, and carboxyl-terminal alpha-subunit synthetic peptides. An anti-Gi1-2 alpha antibody and a Gi2 alpha peptide (Gi2 alpha) Ile345-Phe355), but not a Gi3 alpha peptide (Gi3 alpha Ile344-Phe354), inhibited vasopressin- and thapsigargin-stimulated Ca2+ inflow, had no effect on vasopressin-stimulated release of Ca2+ from intracellular stores, and caused partial inhibition of thapsigargin-stimulated release of Ca2+. An anti-Gq alpha antibody also inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. Immunofluorescence measurements showed that Gi2 alpha is distributed throughout much of the interior of the hepatocyte as well as at the periphery of the cell. By contrast, Gq/11 alpha was found principally at the cell periphery. It is concluded that the trimeric G-protein, Gi2, is required for store-activated Ca2+ inflow in hepatocytes and acts between the release of Ca2+ from the endoplasmic reticulum (presumably adjacent to the plasma membrane) and the receptor-activated Ca2+ channel protein(s) in the plasma membrane.

Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta-adrenoceptor agonists.

Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 microM salbutamol, a beta-adrenoceptor agonist, and 30 microM rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to approximately 125 kDa (PDE4A) and approximately 70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E2 (PGE2), an agent that activates adenylyl cyclase by a non-beta-adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE2-stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, up-regulation of PDE4 markedly decreased the ability of PGE2 to inhibit LTD4-induced Ca2+ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering beta-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous anti-inflammatory agents such as PGE2.

Characterization of Two Activated Mutants of Human pp60c-src That Escape c-Src Kinase Regulation by Distinct Mechanisms

Two activated transforming mutants of human pp60c-src were found to possess single point mutations within the regulatory carboxyl terminus (E527K in CY CST201) and the kinase domain (E381G in WO CST1), respectively, that do not directly interfere with either the regulatory c-Src kinase (CSK) phosphorylation site (Tyr530) or the SH2/3 domains. In vivo, both mutant proteins are hypophosphorylated on their carboxyl-terminal regulatory tyrosines and are hyperactive. In an in vitro Src kinase inactivation assay, both mutant Src proteins exhibited resistance to inactivation by CSK relative to wild-type Src. Under these in vitro conditions, E381G c-Src was found to be phosphorylated by CSK to wild-type levels, while E527K c-Src was not detectably phosphorylated. The ability of CSK to phosphorylate a carboxyl-terminal peptide modelled against E527K c-Src was also impaired, suggesting that CSK is unable to recognize E527K c-Src as an efficient substrate. In the case of E381G c-Src, examination of whether its SH2/3 domains were accessible to the carboxyl-terminal regulatory phosphotyrosine revealed a highly reduced ability of autophosphorylated E381G c-Src to bind to a synthetic phosphopeptide modelled from the SH2-binding region of polyoma middle-T antigen which binds to Src SH2 with high affinity. This suggests that the E381G c-Src mutation results in an altered or reduced accessibility of the SH2 domain of the autophosphorylated form of E381G c-Src and may represent a previously undescribed mode of Src activation. Further study of these and other Src mutants may offer additional new insights into the regulation of "Src family" kinases.

Localization of the Insulin-like Growth Factor I Receptor Binding Sites for the SH2 Domain Proteins p85, Syp, and GTPase Activating Protein

Potential signaling substrates for the insulin-like growth factor I (IGF-I) receptor are SH2 domain proteins including the p85 subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp, GTPase activating protein (GAP), and phospholipase C-gamma (PLC-gamma). In this study, we demonstrate an association between the IGF-I receptor and p85, Syp, and GAP, but not with PLC-gamma in lysates of cells overexpressing the human IGF-I receptor. We further investigated these interactions using glutathione S-transferase (GST) fusion proteins containing the amino-terminal SH2 domains of p85 or GAP, or both SH2 domains of Syp or PLC-gamma to precipitate the IGF-I receptor from purified receptor preparations and from whole cell lysates. p85-, Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the PLC-gamma-GST did not. Using phosphopeptides corresponding to IGF-I receptor phosphorylation sites, we determined that the p85- and Syp-GST association with the IGF-I receptor could be inhibited by a carboxyl-terminal peptide containing pY1316 and that the GAP-GST association could be inhibited by a NPXY domain peptide. The GAP-GST binding site was confirmed by showing that a mutant IGF-I receptor with a deletion of the NPXY domain including tyrosine 950 was poorly precipitated by the GAP-GST. We conclude that p85 and Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that GAP may bind to the IGF-I receptor at and PLC-gamma was not evident. p85, Syp, and GAP are potential modulators of IGF-I receptor signal transduction.