Carboxyl-terminal Truncation Mutants Research Articles

The Carboxyl-terminal PDZ Ligand Motif of Chemokine Receptor CXCR2 Modulates Post-endocytic Sorting and Cellular Chemotaxis

Adaptor protein interaction with specific peptide motifs found within the intracellular, carboxyl terminus of chemokine receptor CXCR2 has been shown to modulate intracellular trafficking and receptor function. Efficient ligand-induced internalization of this receptor is dependent on the binding of adaptor protein 2 to the specific LLKIL motif found within the carboxyl terminus (1). In this study we show that the carboxyl-terminal type 1 PDZ ligand motif (-STTL) of CXCR2 plays an essential role in both proper intracellular receptor trafficking and efficient cellular chemotaxis. First, we show that CXCR2 is sorted to and degraded in the lysosome upon long-term ligand stimulation. We also show that receptor degradation is not dependent upon receptor ubiquitination, but is instead modulated by the carboxyl-terminal type I PDZ ligand of CXCR2. Deletion of this ligand results in increased degradation, earlier co-localization with the lysosome, and enhanced sorting to the Rab7-positive late endosome. We also show that deletion of this ligand effects neither receptor internalization nor receptor recycling. Furthermore, we demonstrate that deletion of the PDZ ligand motif results in impaired chemotactic response. The data presented here demonstrate that the type I PDZ ligand of CXCR2 acts to both delay lysosomal sorting and facilitate proper chemotactic response.

Characterization of the pH-dependent Interaction between the Gap Junction Protein Connexin43 Carboxyl Terminus and Cytoplasmic Loop Domains

A prevailing view regarding the regulation of connexin43 (Cx43) gap junction channels is that, upon intracellular acidification, the carboxyl-terminal domain (Cx43CT) moves toward the channel opening to interact with specific residues acting as a receptor site. Previous studies have demonstrated a direct, pH-dependent interaction between the Cx43CT and a Cx43 cytoplasmic loop (Cx43CL) peptide. This interaction was dependent on alpha-helical formation for the peptide in response to acidification; more recent studies have shown that acidification also induces Cx43CT dimerization. Whether Cx43CT dimerization is an important structural component in Cx43 regulation remains to be determined. Here we used an assortment of complimentary biophysical techniques to characterize the binding of Cx43CT or its mutants to itself and/or to a more native-like Cx43CL construct (Cx43CL(100-155), residues 100-155). Our studies expand the observation that specific Cx43CT domains are important for dimerization. We further show that properties of the Cx43CL(100-155) are different from those of the Cx43CL peptide; solvent acidification leads to Cx43CL(100-155) oligomerization and a change in the stoichiometry and binding affinity for the Cx43CT. Homo-Cx43CT and Cx43CL(100-155) oligomerization as well as the Cx43CT/Cx43CL(100-155) interaction can occur under in vivo conditions; moreover, we show that Cx43CL(100-155) strongly affects resonance peaks corresponding to Cx43CT residues Arg-376-Asp-379 and Asn-343-Lys-346. Overall, our data indicate that many of the sites involved in Cx43CT dimerization are also involved in the Cx43CT/Cx43CL interaction; we further propose that chemically induced Cx43CT and Cx43CL oligomerization is important for the interaction between these cytoplasmic domains, which leads to chemically induced gating of Cx43 channels.

A Structural Determinant of Human Cytomegalovirus US2 Dictates the Down-regulation of Class I Major Histocompatibility Molecules

Human cytomegalovirus down-regulates cell surface class I major histocompatibility (MHC) molecules, thus allowing the virus to proliferate while avoiding detection by CD8+ T lymphocytes. The unique short gene product US2 is a 199-amino acid type I endoplasmic reticulum glycoprotein that modulates surface expression of class I MHC products by targeting class I heavy chains for dislocation from the endoplasmic reticulum to the cytosol, where they undergo proteasomal degradation. Although the mechanism by which this viral protein targets class I heavy chains for destruction remains unclear, the putative US2 cytoplasmic tail comprised of only 14 residues is known to play a functional role. To determine the specific residues critical for mediating class I degradation, a mutagenesis analysis of the cytoplasmic tail of US2 was performed. Using truncation mutants, the removal of only 4 residues (mutant US2(195)) from the US2 carboxyl terminus completely abolishes class I destruction. Furthermore, site-directed mutagenesis of the US2 cytoplasmic tail revealed that the most critical residues for class I-induced destruction, cysteine 187, serine 190, tryptophan 193, and phenylalanine 196, occurs every third residue. This experimental data supports a model that the US2 cytoplasmic tail is in a 3(10) helical configuration. Such a secondary structure would predict that one side of the 3(10) helical cytoplasmic tail would interact with the extraction apparatus to facilitate the dislocation and subsequent destruction of class I heavy chains.

Ubiquitin-dependent Degradation of Interferon Regulatory Factor-8 Mediated by Cbl Down-regulates Interleukin-12 Expression

Interferon regulatory factor (IRF)-8/interferon consensus sequence-binding protein is regulated by both transcription and degradation. IRF-8 induced in peritoneal macrophages by interferon-gamma and lipopolysaccharide was degraded rapidly, and degradation of IRF-8 was blocked by MG132, the proteasome inhibitor, but inhibitors of calpain and lysosomal enzymes had no effect. The ubiquitination of IRF-8 was shown by co-immunoprecipitation from RAW264.7 macrophages retrovirally transduced with IRF-8 and hemagglutinin-ubiquitin. The dominant negative ubiquitin mutants K48R and K29R inhibited IRF-8 degradation in 293T cells, confirming the relationship between ubiquitination of IRF-8 and its degradation. IRF-8 carboxyl-terminal truncation mutants were not ubiquitinated and were consequently stable, indicating that the carboxyl-terminal domain of IRF-8 controls ubiquitination. The ubiquitin-protein isopeptide ligase (E3) that ubiquitinated IRF-8 was likely to be Cbl, which formed a complex with IRF-8, demonstrable by both immunoprecipitation and gel filtration. Furthermore, IRF-8 stability was increased by dominant negative Cbl, and IRF-8 ubiquitination was significantly attenuated in Cbl-/- cells. Reflecting increased stability and expression, the IRF-8 carboxyl-terminal deletion mutant induced interleukin (IL)-12 p40 promoter activity much more strongly than IRF-8 did. Furthermore, IRF-8-induced IL-12 p40 synthesis in RAW264.7 cells was enhanced by dominant negative Cbl, and peritoneal macrophages from Cbl-/- mice showed increased IL-12 p40 protein production. Taken together, these results suggest that the proteasomal degradation of IRF-8 mediated by the ubiquitin E3 ligase Cbl down-regulates IL-12 expression.

TGF‐β Signaling in Colon Cancer Cells

Transforming growth factor-betas (TGF-betas), cytokines expressed in the colon, play important roles as tumor suppressors and tumor promoters during colorectal carcinogenesis. TGF-beta signaling pathway involves activation of Smad2 and Smad3 by the type I receptor and formation of Smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. Most human colorectal cancers are resistant to the tumor suppressor effects of TGF-beta, and a subset of human colorectal cancers have mutations in Smad2 and Smad4. The purpose of this study was to determine whether Smads are required for TGF-beta signaling in colon cancer cells. First, we selected a colon cancer cell line (MC-26) that has a functional TGF-beta signaling pathway. We found that MC-26 cells expressed Smad2, Smad3, and Smad4 mRNAs by reverse transeription-polymerase chain reaction and confirmed that the TGF-beta signaling pathway is functional using a transient transfection assay with 3TP-Lux reporter plasmid. TGF-beta also inhibited cell growth and induced apoptosis in MC-26 cells. When MC-26 cells were transiently transfected with dominant-negative carboxyl-terminal truncation mutants of Smad2, Smad3, and Smad4, TGF-beta-induced 3TP-Lux reporter activity was significantly reduced, suggesting that Smad2, Smad3, and Smad4 are attractive novel therapeutic targets for regulating TGF-beta signaling in colorectal cancers. Because MC-26 cells express TGF-beta activated Smads, have a functional TGF-beta signaling pathway, and are sensitive to the growth inhibitory and apoptotic effects of TGF-beta, they can serve as an excellent model to examine TGF-beta signaling in colorectal cancers.

Casein Kinase Iϵ Plays a Functional Role in the Transforming Growth Factor-β Signaling Pathway

The transforming growth factor-beta (TGF-beta) signaling pathway is known to be involved in a wide range of biological events, including development, cellular differentiation, apoptosis, and oncogenesis. The TGF-beta signal is mediated by ligand binding to the type II receptor, leading to the recruitment and activation of the type I receptor, and subsequent activation of a family of intracellular signal transducing proteins called Smads. Here we report a regulatory role for casein kinase Iepsilon (CKIepsilon) in the TGF-beta signaling cascade. We find that CKIepsilon binds to all Smads and the cytoplasmic domains of the type I and type II receptors both in vitro and in vivo. The interaction of CKIepsilon with the type I and type II receptors is independent of TGF-beta stimulation, whereas the CKIepsilon/Smad interaction is transiently disrupted by ligand treatment. Additionally, CKIepsilon is able to phosphorylate the receptor-activated Smads (Smads 1-3 and 5) and the type II receptor in vitro. Transcriptional reporter assays reveal that transient overexpression of wild type CKIepsilon dramatically reduces basal reporter activity but enhances TGF-beta-stimulated transcription. Furthermore, overexpression of a kinase-dead mutant of CKIepsilon inhibits both basal and ligand-induced transcription, whereas inhibition of endogenous CKI catalytic activity with IC261 blocks only TGF-beta-stimulated reporter activity. Finally, knocking down CKIepsilon protein levels results in a significant increase in basal and TGF-beta-induced transcription. These results suggest that CKIepsilon plays a ligand-dependent, differential, and dual regulatory role within the TGF-beta signaling pathway.

HspBP1, an Hsp70 Cochaperone, Has Two Structural Domains and Is Capable of Altering the Conformation of the Hsp70 ATPase Domain

We present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding.

Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin.

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.

Dendrite-like Process Formation and Cytoskeletal Remodeling Regulated by δ-Catenin Expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of δ-catenin (or NPRAP/Neurojungin), a member of p120ctn subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of δ-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. δ-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of δ-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When δ-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that δ-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.

Deletion of RAR Carboxyl Terminus Reveals Promoter- and Receptor-Specific AF-1 Effects

Retinoic acid receptors (RARs) are transcription factors with both amino-terminal ligand-independent and carboxyl-terminal ligand-dependent activation functions (AF-1 and AF-2, respectively). RAR-dependent gene activation in keratinocytes was investigated via expression of varied RARα and RARγ carboxyl terminal truncation mutants lacking the AF-2 domain. Overexpression of the AF-1 domain of RARα or RARγ was sufficient to decrease transcriptional activation of retinoid-dependent genes in keratinocytes. Conversely, expression of the same constructs was associated with an increase in expression of endogenous and synthetic reporter genes otherwise negatively regulated by RARs. These effects on transcription driven by some but not all retinoid-sensitive promoters tested could be alleviated by mutation of a serine phosphorylation site in the A/B domain. These results further support the promoter-specificity previously attributed to the RAR AF-1 region and functionally define a particular amino acid residue likely to contribute to the regulation of RARs and other proteins in the transcription complex.

Effects of Carboxyl-terminal Truncations on the Activity and Solubility of Human Monoamine Oxidase B

Monoamine oxidase is an outer mitochondrial membrane protein that catalyzes the deamination of a number of neurotransmitters and dietary amines. To determine the roles of the carboxyl-terminal amino acids on the activity and solubility of human monoamine oxidase (MAO B), 10 sequential mutants were made with stop codons at amino acid positions 511, 504, 498, 492, 486, 481, 476, 467, 417, and 397, respectively. All truncated mutants were expressed in Sf21 insect cells using baculovirus, and the enzyme kinetic parameters were determined. Truncations at amino acid positions 511, 504, and 498 slightly decreased MAO B catalytic activity and had no significant changes on deprenyl inhibition. Further deletions up to amino acid 417 decreased the specific activity 10--100-fold without significant changes of the K(m) for phenylethylamine or dopamine or the IC(50) for deprenyl and clorgyline. The truncation mutant C397, which lacks covalently attached FAD, was inactive. Progressive carboxyl-terminal truncations up to position 481 were correlated with increased solubility of MAO B mutants. 47% of the activity of the truncated C481 was found in the 105,000 x g supernatant in the absence of detergent. However, further truncated mutants, C476, C467, and C417, remained associated with the membrane fraction. In contrast to crude homogenate, the water-soluble C481 mutant was rapidly inactivated at 4 degrees C and 37 degrees C, which indicates that the membrane environment is required for the stability of MAO B. Expression of the green fluorescent protein-MAO B C481 fusion protein revealed that this mutant was located in the cytoplasm, whereas its counterpart in MAO A, truncated mutant C490, was located on the mitochondria. These results suggest that the carboxyl-terminal amino acid residues 417--520 of MAO B are not directly involved in the active site but are required for maintaining the appropriate conformation and interaction with the outer mitochondrial membrane. The different solubilities of the various carboxyl-terminal truncation mutants indicate that the interaction of MAO B with mitochondrial membrane is not simply anchoring through the carboxyl-terminal hydrophobic tail. Further, our results suggest that the carboxyl-terminal of MAO A and B plays different roles in mitochondrial attachment.

Inhibition of H-Ras transformation by the PTEN/MMAC1/TEP1 tumor suppressor gene.

The human PTEN/MMAC1/TEP1 (PTEN) tumor suppressor gene encodes a phosphatase with specificity towards the D3 phosphate of phosphatidylinositides. PTEN mutations have been reported in the endometrioid type of uterine tumors which are associated with frequent activations of the Ras oncogenes. In this study, we report the ability of PTEN to potently inhibit H-Ras induced morphological transformation and anchorage-independent growth in NIH3T3 cells. This novel activity of PTEN was correlated more with its ability to suppress the phosphatidylinositol 3-kinase (PI3-K)-dependent signaling cascade, but not the mitogen-activated protein kinase (MAPK) pathway. To define the minimal region in PTEN protein that is responsible for this anti-oncogenic activity, a panel of carboxyl-terminal truncation mutants was generated. While deletions of 4 and 33 amino acids do not have marked effects, removal of up to 68 amino acids drastically reduced the ability of PTEN to inhibit Ras transformation. The propensity of these mutants to suppress Ras transformation is correlated with their relative ability to dephosphorylate inositol (1,3,4,5)-tetrakisphosphate in vitro, and to suppress Akt kinase activity in cultured cells. In addition, we have evidence to suggest that the C-terminal region of PTEN contributes to the stability of the encoded gene product.

The Carboxyl-terminal Domains of gp130-related Cytokine Receptors Are Necessary for Suppressing Embryonic Stem Cell Differentiation: INVOLVEMENT OF STAT3

Cell type-specific responses to the leukemia inhibitory factor (LIF)/interleukin 6 cytokine family are mediated by dimerization of the LIF receptor alpha-chain (LIFRalpha) with the signal transducer gp130 or of two gp130 molecules followed by activation of the JAK/STAT and Ras/mitogen-activated protein kinase cascades. In order to dissect the contribution of gp130 and LIFRalpha individually, chimeric molecules consisting of the extracellular domain of the granulocyte colony stimulating factor receptor (GCSF-R) and various mutant forms of the cytoplasmic domains of gp130 or LIFRalpha were expressed in embryonic stem (ES) cells to test for suppression of differentiation, or in a factor-dependent plasma cytoma cell line to assess for induction of proliferation. Carboxyl-terminal domains downstream of the phosphatase (SHP2)-binding sites were dispensable for mitogen-activated protein kinase activation and the transduction of proliferative signals. Moreover, carboxyl-terminal truncation mutants which lacked intact Box 3 homology domains showed decreased STAT3 activation, failed to induce Hck kinase activity and suppress ES cell differentiation. Moreover, STAT3 antisense oligonucleotides impaired LIF-dependent inhibition of differentiation. Substitution of the tyrosine residue within the Box 3 region of the GSCF-R abolished receptor-mediated suppression of differentiation without affecting the transduction of proliferative signals. Thus, distinct cytoplasmic domains within the LIFRalpha, gp130, and GCSF-R transduce proliferative and differentiation suppressing signals.

Agonist and antagonists induce homodimerization and mixed ligand heterodimerization of human progesterone receptors in vivo by a mammalian two-hybrid assay.

This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4-13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two-hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist. Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins. However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur. Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity. Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR. We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.

Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control.

The cyclin-dependent kinase inhibitor p21(Cip1/WAF1) has been implicated as an inducer of differentiation. However, although expression of p21 is increased in postmitotic cells immediately adjacent to the proliferative compartment, its expression is decreased in cells further along the differentiation program. Expression of the p21 protein was decreased in terminally differentiated primary keratinocytes of mice, and this occurred by a proteasome-dependent pathway. Forced expression of p21 in these cells inhibited the expression of markers of terminal differentiation at both the protein and messenger RNA levels. These inhibitory effects on differentiation were not observed with a carboxyl-terminal truncation mutant or with the unrelated cyclin-dependent kinase inhibitor p16(INK4a), although all these molecules exerted similar inhibition of cell growth. These findings reveal an inhibitory role of p21 in the late stages of differentiation that does not result from the effects of p21 on the cell cycle.

Ultrastructural studies of carboxyl-terminal truncation mutants of the neuronal intermediate filament protein peripherin.

We have prepared carboxyl-terminal truncation mutants of the neuronal intermediate filament (IF) protein peripherin and examined the assembly characteristics of these mutant proteins in SW13 cells in the presence and absence of vimentin. In the absence of vimentin, tailless peripherin protein (Per-C424) self-assembles into bundles and clumps as observed by immunofluorescence, whereas a peripherin mutant that is missing the tail as well as a small portion of the rod (Per-C356) appears as spherical aggregates. Similar phenotypes are observed when vimentin-positive cells are transfected with Per-C424 or Per-C356. In these cells, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundles and clumps formed by Per-C424 at the electron microscopic level, we prepared stable cell lines expressing different levels of this mutant protein. By immunofluorescence, Per-C424 appears as either clumps or bundles of filaments depending on the expression level of the mutant protein. However, under electron microscopy, it is apparent that both clumps and bundles are composed of tightly packed IFs. We were unable to obtain stable cell lines expressing Per-C356, indicating that this mutant may prevent cell proliferation. Using a vector containing an internal ribosomal entry site, we prepared a construct that expresses Per-C356 and green fluorescent protein as a single mRNA, and we were able to isolate cells that expressed Per-C356 by fluorescence-activated cell sorting. Electron microscopic analysis of these cells showed that these aggregates are solid and contain no obvious filamentous structures.

Ras complements the carboxyl terminus of v-Abl protein in lymphoid transformation.

Abelson murine leukemia virus (Ab-MLV) mutants expressing v-Abl proteins lacking the carboxyl terminus are compromised in the ability to transform lymphoid but not NIH 3T3 cells. This feature correlates with the presence of low levels of phosphotyrosine in lymphoid cells infected with carboxyl-terminal truncation mutants. In contrast, high levels of phosphotyrosine are observed in NIH 3T3 cells infected with wild-type and mutant Ab-MLV. Two downstream targets affected in lymphoid transformants are the GTPase-activating protein and GTPase-activating protein-associated protein p62, molecules which are heavily tyrosine phosphorylated in lymphoid cells transformed by wild-type Ab-MLV but not carboxyl-terminal truncation mutants of Ab-MLV. This difference suggested that signaling mediated via the Ras pathway may be compromised in lymphoid cells expressing the carboxyl-terminal truncation mutants. Consistent with this idea, expression of v-Ha-ras complemented these mutants in primary bone marrow transformation assays and increased transformation frequencies obtained with the Ab-MLV mutants 8- to 20-fold. These data suggest that a biologically important link exists between the carboxyl terminus of v-Abl protein and the Ras pathway. Signals transmitted via this connection may enhance those mediated via other regions of the v-Abl protein and facilitate transformation of primary, nonimmortalized cells such as pre-B lymphocytes.

Agonist-stimulated phosphorylation of the carboxyl-terminal tail of the secretin receptor: [formula omitted], M. Holtmann, R. Rentsch, L.J. Miller. GI Basic Research Unit, Mayo Clinic, Rochester, MN 55905

The secretin receptor belongs to a recently recognized family of G protein-coupled receptors that lack the sequence motifs typical of the beta-adrenergic receptor family. Because our understanding of the regulatory mechanisms for these receptors is largely based on the latter group, we have begun to explore these mechanisms in the secretin receptor. In the present study, we focused on receptor phosphorylation, a key mechanism of receptor desensitization. Secretin receptor phosphorylation was demonstrated in intact transiently transfected COS cells and a stable receptor-bearing Chinese hamster ovary cell line in response to stimulation with native agonist. Secretin phosphoreceptor migrated on a sodium dodecyl sulfate-polyacrylamide gel at M(r) 57,000-62,000 in its native state and at M(r) 42,000 after deglycosylation, similar to the receptor that had been affinity-labeled with 125I-[Tyr10,p-NO2-Phe22]-secretin-27. Phosphorylation occurred rapidly in a secretagogue concentration-dependent manner, with 0.1 microM secretin eliciting a 7.2-fold increase in phosphorylation after 2 min. One-dimensional phosphopeptide mapping after cyanogen bromide cleavage revealed a single band of M(r) 9400, corresponding in size to the carboxyl-terminal tail domain. This identification was confirmed with a truncation mutant in which potential sites of phosphorylation in the tail were eliminated and no agonist-stimulated phosphorylation was observed. Phosphoamino acid analysis of the secretin phosphoreceptor demonstrated predominance of phosphothreonine over phosphoserine (3.2:1), with no phosphotyrosine observed. Three distinct carboxyl-terminal truncation mutants were constructed to each eliminate a subset of potential phosphorylation sites, and differential levels of phosphorylation were observed. Appropriate biosynthetic processing, expression on the cell surface, and signaling for each of these constructs were ensured by demonstration of ligand binding and cAMP responsiveness. Thus, receptors in the recently described secretin receptor family are phosphorylated in response to agonist stimulation in a manner analogous to the beta-adrenergic receptor, likely representing an important molecular mechanism for receptor desensitization.

The effect of H pylori eradication on symptom relief in patients with functional dyspepsia

• AGONIST-STIMULATED PHOSPHORYLATION OF THE CARBOXYLTERMINAL TAIL OF THE SECRETIN RECEPTOR. F. 0zcelebi, M. Holtmann, R. Rentsch, L.J. Miller. GI Basic Research Unit, Mayo Clinic, Rochester, MN 55905 The secretin receptor belongs to a distinct family of receptors in the G protein-coupled receptor superfamily. The absence of sequence motifs typical of the B-adrenergic receptor family in which regulatory mechanisms have been extensively studied, raises questions of the comparability of regulation of the secretin receptor family. In this work, we have focused on a key mechanism of receptor desensitization, receptor phosphorylation, in cell fines expressing the rat secretin receptor. We have studied receptor phosphorylation in intact transiently transfected COS cells and a stable receptor-bearing CHO cell line, utilizing a strategy we described for the acinar cell CCK receptor (Klueppelberg et al, J. Biol. Chem. 1991). Indeed, this approach resulted in the demonstration of agonist-stimulated receptor phosphorylation. Secretin phosphoreceptor migrated on a SDS-polyacrylamide gel at M,=57,000-62,000 in its native state, and at M,=42,000 after deglycosylation. This is identical to electrophoretic behavior of the affinity-labeled receptor. Phosphorylation occurred rapidly in a secretagogue concentration-dependent manner, with I#M secretin eliciting a 7.2fold increase in phosphorylation after 2 min. One-dimensional phosphopeptide mapping after cyanogen bromide cleavage revealed a single phosphopeptide band of approximate M,= 9,400, corresponding to the expected size of the carboxylterminal tail of the receptor. This identification was then confirmed with a truncation mutant in which all potential sites of phosphorylation in that domain were eliminated, and no agonist-stimulated phosphorylation was observed. Phosphoamino acid analysis of the secretin phosphoreceptor demonstrated predominance of phosphothreonine over phosphoserine (3.2:1), with no phosphotyrosine observed. Three distinct carboxyl-terminal truncation mutants were constructed to each eliminate a subset of potential phosphorylation sites. Appropriate biosynthetic processing, expression on the cell surface, ligand binding, and signalling were assured by demonstration of affinity labeling and cAMP responsiveness. Differential levels of phosphorylation provide additional insights into the distinct residues phosphorylated on this receptor molecule. In conclusion, this work demonstrates for the first time that receptors in the newlydescribed secretin receptor family are phosphorylated in response to agonist stimulation. The molecular details of secretin receptor phosphorylation are highly analogous to those of the /3-adrenergic receptor family. This suggests that phosphorylation is also likely to represent an important molecular mechanism for receptor desensitization for this family of receptors. THE EFFECT OF H PYLOR[ ERADICATION ON SYMPTOM RELIEF IN PATIENTS WITH FUNCTIONAL DYSPEPSIA. MM Ozmen, RS Patankar, CD Johnson

Carboxyl-terminal Domain Truncation Alters Apolipoprotein A-I in Vivo Catabolism

Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver. To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis. ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage. The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I. The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day). All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3. Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I. These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.