Studies on the muscle-specific regulatory protein myogenin

Recently, a family of muscle-specific regulatory factors that includes myogenin, myoD, myf-5, and MRF-4 has been identified. They share a high degree of homology within a region that contains a basic and helix-loop-helix domain. Transfection of many non-muscle cell types with any one of these genes results in the activation of the entire myogenic program. To explore the mechanism through which myogenin regulates myogenesis, we have prepared antibodies against peptides specific to myogenin. Using these antibodies we show that myogenin is a 32 Kd phospho-protein which is localized to the nuclei of muscle cells. In vitro, myogenin oligomerizes with the ubiquitous enhancer binding factor E12, and acquires high affinity for an element of the core of the muscle creatine kinase (MCK) enhancer that is conserved among many muscle-specific genes. Myogenin synthesized in BC$\sb3$H1 and C2 muscle cell lines also binds to the same site in the enhancer. However, the MCK enhancer is not activated in 10T1/2 fibroblasts which have been transfected with a constitutive myogenin expression vector until growth factors have been removed from the media. This result indicates that mitogenic signals block the actions of myogenin.. Mutagenesis of the myogenin/E12 binding site in the MCK enhancer abolishes binding of the hetero-oligomer and prevents trans-activation of the enhancer by myogenin. By site directed mutagenesis of myogenin we have shown that the basic region consists of three clusters of basic residues, two of which are required for binding and activation of the myogenic program. Myogenic activation, but not DNA binding, is lost when the 10 residue region between the two required basic clusters is substituted with the corresponding region from E12, which also contains a similar basic and helix-loop-helix domain. Functional revertants of this substitution mutant have identified two amino acids which confer muscle specificity. The properties of myogenin suggest that it functions as a sequence-specific DNA binding factor that interacts directly with muscle-specific genes during myogenesis and contains within its basic domain a region which imparts myogenic activation and is separable from DNA binding. ^

The Immune Inhibitor A1 Protease of Bacillus anthracis

Bacillus anthracis, an organism ubiquitous in the soil and the causative agent of anthrax, utilizes multiple mechanisms to regulate secreted factors; one example is the activity of secreted proteases. One of the most abundant proteins in the culture supernates of B. anthracis is the Immune Inhibitor A1 (InhA1) protease. Here, I demonstrate that InhA1 modulates the abundance of approximately half of the proteins secreted into the culture supernates, including substrates that are known to contribute to the ability of the organism to cause virulence. For example, InhA1 cleaves the anthrax toxin proteins, PA, LF, and EF. InhA1 also targets a number of additional proteases, including Npr599, contributing to a complex proteolytic regulatory cascade with far-reaching affects on the secretome. Using an intra-tracheal mouse model of infection, I found that an inhA-null strain is attenuated in relation to the parent strain. The data indicate that reduced virulence of the inhA mutant strain may be the result of toxin protein deregulation, decreased association with macrophages, and/or the inability to degrade host antimicrobial peptides. Given the significant modulation of the secretome by InhA1, it is likely that expression of the protease is tightly regulated. To test this I examined inhA1 transcript and protein levels in the parent and various isogenic mutant strains and found that InhA1 expression is regulated by several mechanisms. First, the steady state levels of inhA1 transcript are controlled by the regulatory protein SinR, which inhibits inhA1 expression. Second, InhA1 abundance is inversely proportional to the SinR-regulated protease camelysin, indicating the post-transcriptional regulation of InhA1 by camelysin. Third, InhA1 activity is dependent on a conserved zinc binding motif, suggesting that zinc availability regulates InhA1 activity. The convergence of these regulatory mechanisms signifies the importance of tight regulation of InhA1 activity, activity that substantially affects how B. anthracis interacts with its environment.

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

Genetic studies of the human plasma orosomucoid (alpha-1 acid glycoprotein)

Orosomucoid (ORM) or alpha-1 acid glycoprotein is an acute phase protein of human plasma whose function is suggested to be the competitive inhibition of cellular recognition by infective agents. Isoelectric focusing (IEF) and immunoblotting have been combined and optimum conditions have been determined for reliable classification of different ORM phenotypes. Addition of 6 M urea in an IEF gel revealed additional microheterogeneity in the ORM system which has not been previously reported. 1,667 individuals from different native ethnic groups of North and South America, Africa and New Guinea have been screened to determine the distribution of ORM alleles. Two common alleles, ORM1*1 and ORM1*2 have been observed and their frequencies were determined. Genetically independent variation consistent with expression of the ORM2 locus was observed in American and African blacks but was not observed in other sampled populations. The population allele frequencies for this new locus were 0.958, 0.025, 0.006, 0.011, for alleles ORM2*1, ORM2*2, ORM2*3, ORM2*4, respectively. Family studies confirm the autosomal codominant inheritance of the phenotypes observed at both ORM loci. ^

The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

From protein microarray to biology: The discovery of epigenetic regulatory molecules

Chromatin, composed of repeating nucleosome units, is the genetic polymer of life. To aid in DNA compaction and organized storage, the double helix wraps around a core complex of histone proteins to form the nucleosome, and is therefore no longer freely accessible to cellular proteins for the processes of transcription, replication and DNA repair. Over the course of evolution, DNA-based applications have developed routes to access DNA bound up in chromatin, and further, have actually utilized the chromatin structure to create another level of complexity and information storage. The histone molecules that DNA surrounds have free-floating tails that extend out of the nucleosome. These tails are post-translationally modified to create docking sites for the proteins involved in transcription, replication and repair, thus providing one prominent way that specific genomic sequences are accessed and manipulated. Adding another degree of information storage, histone tail-modifications paint the genome in precise manners to influence a state of transcriptional activity or repression, to generate euchromatin, containing gene-dense regions, or heterochromatin, containing repeat sequences and low-density gene regions. The work presented here is the study of histone tail modifications, how they are written and how they are read, divided into two projects. Both begin with protein microarray experiments where we discover the protein domains that can bind modified histone tails, and how multiple tail modifications can influence this binding. Project one then looks deeper into the enzymes that lay down the tail modifications. Specifically, we studied histone-tail arginine methylation by PRMT6. We found that methylation of a specific histone residue by PRMT6, arginine 2 of H3, can antagonize the binding of protein domains to the H3 tail and therefore affect transcription of genes regulated by the H3-tail binding proteins. Project two focuses on a protein we identified to bind modified histone tails, PHF20, and was an endeavor to discover the biological role of this protein. Thus, in total, we are looking at a complete process: (1) histone tail modification by an enzyme (here, PRMT6), (2) how this and other modifications are bound by conserved protein domains, and (3) by using PHF20 as an example, the functional outcome of binding through investigating the biological role of a chromatin reader. ^

SIGNALING MECHANISMS THAT CONTROL GAP JUNCTIONAL COUPLING BETWEEN RETINAL NEURONS

Gap junctions between neurons form the structural substrate for electrical synapses. Connexin 36 (Cx36, and its non-mammalian ortholog connexin 35) is the major neuronal gap junction protein in the central nervous system (CNS), and contributes to several important neuronal functions including neuronal synchronization, signal averaging, network oscillations, and motor learning. Connexin 36 is strongly expressed in the retina, where it is an obligatory component of the high-sensitivity rod photoreceptor pathway. A fundamental requirement of the retina is to adapt to broadly varying inputs in order to maintain a dynamic range of signaling output. Modulation of the strength of electrical coupling between networks of retinal neurons, including the Cx36-coupled AII amacrine cell in the primary rod circuit, is a hallmark of retinal luminance adaptation. However, very little is known about the mechanisms regulating dynamic modulation of Cx36-mediated coupling. The primary goal of this work was to understand how cellular signaling mechanisms regulate coupling through Cx36 gap junctions. We began by developing and characterizing phospho-specific antibodies against key regulatory phosphorylation sites on Cx36. Using these tools we showed that phosphorylation of Cx35 in fish models varies with light adaptation state, and is modulated by acute changes in background illumination. We next turned our focus to the well-studied and readily identifiable AII amacrine cell in mammalian retina. Using this model we showed that increased phosphorylation of Cx36 is directly related to increased coupling through these gap junctions, and that the dopamine-stimulated uncoupling of the AII network is mediated by dephosphorylation of Cx36 via protein kinase A-stimulated protein phosphatase 2A activity. We then showed that increased phosphorylation of Cx36 on the AII amacrine network is driven by depolarization of presynaptic ON-type bipolar cells as well as background light increments. This increase in phosphorylation is mediated by activation of extrasynaptic NMDA receptors associated with Cx36 gap junctions on AII amacrine cells and by Ca2+-calmodulin-dependent protein kinase II activation. Finally, these studies indicated that coupling is regulated locally at individual gap junction plaques. This work provides a framework for future study of regulation of Cx36-mediated coupling, in which increased phosphorylation of Cx36 indicates increased neuronal coupling.

Cell Cycle Regulatory Roles of Estrogen Receptor alpha (ERα) in Breast Cancer Cells

Previous studies have shown that Estrogen Receptor alpha (ERα) is an important indicator for diagnosis, prognosis and treatment of breast cancers. However, the question remains as to the role of ERα in the cell in the presence versus absence of 17-β estradiol In this dissertation the role of ERα in both its unliganded and liganded state, with respect to the cell cycle will be explored. The cell line models used in this project are ER-positive MCF-7 cells with and without siRNA to ERα and ER-positive MDA-MB-231 cells that have been engineered to express ERα. Cells were synchronized and the cell cycle progression was monitored by flow cytometric analysis. Using these methods, two specific questions were addressed: Does ERα modulate the cell cycle differently under liganded versus unliganded conditions? And, does the presence of ERα regulate cell cycle phase transitions? The results show for the first time that ERα is cell cycle regulated and modulates the progression of cells through S and G2/M phases of the cell cycle. Ligand bound ERα increases progression through S and G2/M phases, whereas unliganded ERα acts as an inhibitor of cell cycle progression. To further investigate the cell cycle regulated effects of liganded ERα, a luciferase assay was performed and showed that the transcription of target genes such as Progestrone Receptor (PgR) and Trefoil protein (pS2) increased duing S and G2/M phases when ERα is bound to ligand. Additionally, complex formation between cyclin B and ER α was shown by immunoprecipitation and led to the discovery that anaphase promoting complex (APC) is the E3 ligase for both cyclin B and ERα at the termination of M phase. Our findings suggest that unliganded ERα has an inhibitory effect on the progression of the cell cycle. Therefore, it is reasonable to speculate that the combination of drugs that lower estrogen level (such as aromatase inhibitors) and preserves ERα from degradation would provide better outcome for breast cancer treatment. We have shown that APC functions as the E3 ligase for ERα and thus might provide a target to design a specific inhibitor of ERα degradation.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

Isolation and analysis of regulatory and structural mutations in the {\it recA\/} gene of {\it Escherichia coli}

The recA gene is essential for homologous recombination and for inducible DNA repair in Escherichia coli. The level of recA expression is important for these functions. The growth defect of a lambda phage carrying a recA-lacZ fusion was used to select mutations that reduced recA expression. Nine of these mutations were single base changes in the recA promoter; each reduced both induced and basal (repressed) levels of expression, indicating that only one promoter is used under both circumstances. Deletion analysis of the promoter region and S1 mapping of transcripts confirmed that there is only one promoter responsible for both basal and induced expression. Some of the mutants, however, displayed a ratio of induced to repressed expression that was much lower than wild-type. For one of these mutants (recA1270) LexA binding studies showed that this was not due to a change in the affinity of LexA repressor for the operator site. The extent of binding of RNA polymerase to this mutant promoter, however, was much reduced, and the complexes formed were qualitatively different. Further binding experiments provided some evidence that LexA does not block RNA polymerase binding to the recA promoter, but inhibits a later step in initiation. Behavior of the mutants with altered induction ratios could be explained if LexA binding to the operator actually increases RNA polymerase binding to the promoter in a closed complex compensating for defects in polymerase binding caused by the mutations.^ In a study of mutations in the recA structural gene, site-directed mutagenesis was used to replace cysteine codons at positions 90, 116, and 129 with a number of different codons. In vivo analysis of the replacements showed that none of the cysteines is absolutely essential and that they do not have a direct role as catalysts in ATP hydrolysis. Some amino acid substitutions abolished all RecA functions, while a few resulted in partial or altered function. Amino acids at positions 90 and 129 tended to affect all functions equally, while the amino acid at position 116 appeared to have a particular effect on the protease activity of the protein. ^

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Fatty acylated proteins and the protein kinase C signaling pathway

Numerous proteins in intracellular signaling pathways are known to be covalently modified by long chain fatty acids. The objective of this project was to identify potentially novel components of the protein kinase C signaling pathway by virtue of their fatty acylation. A 64 kDa palmitoylated protein (p64) was identified that became deacylated following stimulation of quiescent cells with serum, FGF, or PDBu, suggesting that stimulus-dependent deacylation might alter interactions between p64 and other membrane/cytoskeletal components. A myristoylated protein of 68 kDa observed during these studies was identified as the "80K" PKC substrate. This protein was acylated cotranslationally with myristate through an amide linkage. The majority of the 80K protein was tightly associated with the plasma membrane, with approximately 20% in the cytosol. Although phosphorylation of the membrane-bound and soluble forms of the protein was increased 6-fold in response to PDBu, no changes in the subcellular distribution or myristoylation of the protein were observed. A cDNA encoding the murine form of this protein was cloned, and its deduced amino acid sequence revealed the presence of an N-terminal myristoylation consensus and five potential sites for phosphorylation by PKC. A mutant in which the N-terminal glycine residue was changed to alanine was no longer a substrate for NMT and consequently lost its membrane-binding potential. However, its ability to be phosphorylated in response to purified growth factors and phorbol esters was unimpaired. These results indicate that the myristoylated N-terminus of the 80K protein is required for its association with the plasma membrane, and that the cytoplasmic form of the protein can be phosphorylated independently of the membrane-bound form. Mutants of PKC were constructed in which the regulatory domain was removed and replaced by the N-terminus of the 80K or Al proteins. Unexpectedly, both the myristoylated and nonmyristoylated fusion proteins were tightly associated with the nuclear envelope. Further deletion analyses mapped nuclear targeting signals to the hinge region and a portion of the catalytic domain of PKC, explaining the ability of PKC to be translocated to the nucleus in response to certain stimuli. ^

Expression and regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis

Factors involved in regulating tissue specific gene expression play a major role in cell differentiation. In order to further understand the differentiation events occurring during hematopoiesis, a myeloid specific gene was characterized, the expression pattern during hematopoiesis was analyzed, and the mechanisms governing its regulation were assessed. Previously, our laboratory isolated an anonymous cDNA clone, pD-D1, which displayed preferential expression in myeloid cells. From nucleotide sequencing of overlapping cDNA clones I determined that the D-D1 message encodes a hematopoietic proteoglycan core protein (HpPG). The expression pattern of the gene was assessed by in situ hybridization of bone marrow and peripheral blood samples. The gene was shown to be expressed, at variable levels, in all leukocytes analyzed, including cells from every stage of neutrophil development. In an attempt to ascertain the differentiation time point in which the HpPG gene is initially expressed, more immature populations of leukemic myeloblasts were assessed by northern blot analysis. Though the initial point of expression was not obtained, an up-regulatory event was discovered corresponding to a time point in which granule genesis occurs. This finding is consistent with prior observations of extensive packaging of proteoglycans into the secretory granules of granule producing hematopoietic cells. The HpPG gene was also found to be expressed at low levels in all stages of lymphocyte development analyzed, suggesting that the HpPG gene is initially expressed before the decision for myeloid-lymphoid differentiation. To assess the mechanism for the up-regulatory event, a K562 in vitro megakaryocytic differentiation system was used. Nuclear run-off analyses in this system demonstrated the up-regulation to be under transcriptional control. In addition, the HpPG gene was found to be down regulated during macrophage differentiation of HL60 cells and was also shown to be transcriptionally controlled. These results indicate that there are multiple points of transcriptional regulation of the HpPG gene during differentiation. Furthermore, the factors regulating the gene at these time points are likely to play an important role in the differentiation of granule producing cells and macrophages. ^

Study of tumor suppressor genes p53 and Rb in acute myelogenous leukemia

Loss of antiproliferative function of p53 by point mutation occurred frequently in various solid tumors. However, the genetic change of p53 by deletion or point mutation was a rare event (6%) in the cells of 49 AML patients analyzed by single-stranded conformation polymorphism and sequencing. Despite infrequent point mutation, abundant levels of p53 protein were detected in 75% of AML patients studied by immunoprecipitation with p53 specific antibodies. Furthermore, p53 protein in most cases had an altered conformation as analyzed by the reactivity to PAb240 which recognizes mutant p53; p53 protein in mitogen stimulated normal lymphocytes also had similar altered conformation. This altered conformation may be another mechanism for inactivation of p53 function in the growth stimulated environment. Some evidence indicated that posttranslational modification by phosphorylation may contribute to the conformational change of p53.^ Retinoblastoma (Rb) gene inactivation by deletion, rearrangement or mutation has also been implicated in many types of solid tumors. Our studies showed that absence or low levels of Rb protein were observed in more than 20% of AML patients at diagnosis, and the low levels of Rb correlated with shorter survival of patients. The absence of Rb protein was due to gene inactivation in some cases and to abnormal regulation of Rb expression in others. ^

Regulation of the gastrin-releasing peptide/bombesin receptor

Gastrin-releasing peptide (GRP) and other bombesin-like peptides stimulate hormone secretion and cell proliferation by binding to specific G-protein-coupled receptors. Three studies were performed to identify potential mechanisms involved in GRP/bombesin receptor regulation.^ Although bombesin receptors are localized throughout the gastrointestinal tract, few gastrointestinal cell lines are available to study bombesin action. In the first study, the binding and function of bombesin receptors in the human HuTu-80 duodenal cancer cell line were characterized. ($\sp{125}$I-Tyr$\sp4$) bombesin bound with high affinity to a GRP-preferring receptor. Bombesin treatment increased IP$\sb3$ production, but had no effect on cell proliferation. Similar processing of ($\sp{125}$I-Tyr$\sp4$) bombesin and of GRP-receptors was observed in HuTu-80 cells and Swiss 3T3 fibroblasts, a cell line which mitogenically responds to bombesin. Therefore, the lack of a bombesin mitogenic effect in HuTu-80 cells is not due to unusual processing of ($\sp{125}$I-Tyr$\sp4$) bombesin or rapid GRP-receptor down-regulation.^ In the second study, a bombesin antagonist was developed to study the processing and regulatory events after antagonist binding. As previously shown, receptor bound agonist, ($\sp{125}$I-Tyr$\sp4$) bombesin, was rapidly internalized and degraded in chloroquine-sensitive compartments. Interestingly, receptor-bound antagonist, ($\sp{125}$I-D-Tyr$\sp6$) bombesin(6-13)PA was not internalized, but degraded at the cell-surface. In contrast to bombesin, (D-Tyr$\sp6$) bombesin(6-13)PA treatment did not cause receptor internalization. Together these results demonstrate that receptor regulation and receptor-mediated processing of antagonist is different from that of agonist.^ Bombesin receptors undergo acute desensitization. By analogy to other G-protein-coupled receptors, a potential desensitization mechanism may involve receptor phosphorylation. In the final study, $\sp{32}$P-labelled Swiss 3T3 fibroblasts and CHO-mBR1 cells were treated with bombesin and the GRP-receptor was immunoprecipitated. In both cell lines, bombesin treatment markedly stimulated GRP-receptor phosphorylation. Furthermore, bombesin-stimulated GRP-receptor phosphorylation occurred within the same time period as bombesin-stimulated desensitization, demonstrating that these two processes are correlated.^ In conclusion, these studies of GRP-receptor regulation further our understanding of bombesin action and provide insight into G-protein-coupled receptor regulation in general. ^