John R. Dedman - US grants

It is well established that Ca2+ is a pleiotrophic factor affecting a wide variety of essential cellular processes. It is postulated that many of these actions are mediated via calmodulin. In fact, it is felt that the Ca2+ calmodulin complex may represent the active cellular state of Ca2+. Since all cells contain calmodulin and Ca2+, the specificity of Ca2+ calmoduling action in cells is somewhat of an enigma. Our results, as well as published results of others suggests that specificity of Ca2+ action resides in the calmodulin acceptor proteins (CAPs). That is, the response of any particular cell type to elevated Ca2+ levels is dependent upon the class and population of CAPs. These proteins represent a variety of enzymes which are regulated by Ca2+ calmodulin. It is very possible that the CAPs themselves possess enzyme modifying properties (i.e. protein kinase and methylases). The tissue specificity of such a situation would be extended to not only the CAPs, but the presence of CAP protein substrate as well. We propose to continue our purification of CAPs from electroplax and chicken gizzard. These studies include the subunit structure, immunolocalization, and expression during different physiological states in hopes of better understanding their individual functional roles. We have recently discovered several other high-affinity calcium receptor proteins which form calcium induced hydrophobicity. Due to their functional similarity with troponin C and calmodulin we have referred to them as "calcimedins". We now have the ability to purify these proteins in high quantity in order to pursue a complete physicochemical characterization, produce antibodies, develop immunoassays, and study their functional role in cell regulation. Collectively, our studies will provide a better understanding regarding the mediation and selective discrimination of the regulatory intracellular calcium signal.

The long-term goal of this research is to obtain a better understanding of the control of cell proliferation at sites beyond the cell membrane. There appears now to be a limited number of cancer-causing genes or "oncogenes." However, our knowledge relating the expression of these gene products with neoplastic transformation is deficient in molecular detail. Calcium and calmodulin play key roles as second messengers in cell growth, division, and differentiation. Neoplastic cells are autonomous to the control properties of calcium and have elevated calmodulin levels. Our group has developed a cadre of expertise to now dissect the calcium-calmodulin pathways leading to transformation. We propose to elevate calmodulin levels by microinjecting pure calmodulin, its mRNA, and its structural gene. We can then test the hypothesis that elevated cellular calmodulin results in loss in growth regulation. Oncomodulin, a low molecular weight tumor-specific protein, will be purified and investigated for its growth-promoting properties. In vivo and in vitro phosphorylation studies will be performed using cell lines temperature-sensitive for transformation in order to determine the role of protein kinase C. This calcium-phospholipid kinase represents the major receptor for active phorbol diesters. An additional aspect of this work is the identification and isolation of the calmodulin acceptor proteins (CAPs) associated with neoplasia. Monoclonal antibodies will be produced and quantitative measurements of expression will be performed during neoplastic transformation. These studies will involve sarcoma-virus-infected temperature-sensitive rat kidney cells. Subcellular localization will be determined using immunofluorescence microscopy. Finally, we propose to functionally characterize the growth-related CAP, calplasin. We have shown calplasin to be expressed only during cell growth in the nucleolus. Using an in vitro system, the effects of calcium, calmodulin, oncomodulin, calplasin, and anticalplasin on ribosomal RNA synthesis and processing will be examined. Collectively, the experiments should provide direct evidence which will couple the calcium signal with growth regulation via specific cellular proteins. (N)

Rat parotid adenylate cyclase and Alpha-amylase secretion desensitize to Beta-adrenergic agonists without any receptor down-regulation. The hypothesis that desensitization of adenylate cyclase activity to Beta-adrenergic agonists in rat parotid is mediated through effects involving the catalytic subunit of adenylate cyclase will be tested. The specific aims of the proposal are to determine the mechanism(s) by which desensitization is produced and by which forskolin counteracts desensitization. Monoclonal antibodies have been raised to purified adenylate cyclase catalytic subunit (protein obtained from Dr. A. G. Gilman). Desensitized and control adenylate cyclase catalytic subunit will be isolated by specific antibody interaction or by affinity chromatography on forskolin-Sepharose. Metabolic labelling studies will be performed to determine whether desensitization is produced by incorporation of a covalent modification into the catalytic subunit, or if a potential Desensitizing Ligand is responsible. Carbachol produces a stable depression of adenylate cyclase in rat parotid which is pharmacologically similar to isoproterenol-produced desensitization. The mechanism of carbachol effect will likewise be determined. Forskolin counteracts desensitization and stable depression, respectively. Its mechanism of action will be tested similarly. Phorbol esters have been found to produce desensitization symptoms, and their mechanism of doing so will be established. Reconstitution of purified components is the ultimate goal. Rat parotid will be used as a model tissue for understanding post-receptor mechanisms of desensitization. Desensitized Beta-adrenergic response is a major problem in therapy of certain disease states, including asthma. Better understanding of physiological control of salivary secretion will also result from these studies.

Cystic fibrosis, an autosomally recessive disease, has long been recognized as a disease of glandular secretion. Affected tissues secrete less water resulting in thick mucus congestion of the lungs, airway passages, pancreas and intestine. The collective works of several laboratories have shown the disease to be a defect in the secretion of chloride ions from epithelial cells. More specifically, it has been shown that cystic fibrosis is a diseae of chloride channel regulation. Normally, this channel is opened in response to the second messenger cyclic AMP and calcium. CF patients show failures to secrete chloride in response to cyclic AMP and in some tissues calcium mediated secretagogues. For the past several years our laboratories have been investigating the mediation of intracellular calcium through the identification of high affinity calcium-binding proteins. Recently a family of calcium/phospholipid-binding proteins had been identified yet specific cellular function has only been implied. We propose to use immunohistochemical techniques to localize thee "calcimedins" in transporting epithelia. In addition subcellular apical and basolateral membrane fractions will be analyzed for calcimedin content, calcium- dependent calcimedin-binding and calcimedin-binding proteins. Regulations of the epithelial chloride channel by the calcimedins will be through reconstitution studies in artificial bilayers, whole-cell patch-clamp and inside-out membrane patches. Efforts will be made to reconstitute tracheal, small intestine and rectal gland chloride channels in order to delineate the regulatory elements which may include one of the calcimedins. One means of pharmacologically treating cystic fibrosis would be to activate a calcium mediator causing he channel to open and potentially ameliorate the chronic condition.

Changes in intracellular calcium represent an essential signal in excitation contraction and stimulus-response coupling. The objective of our laboratory has been directed towards identifying intracellular calcium-binding proteins in order to understand the mediation of the intracellular calcium signal. This revised renewal is focused towards elucidating the physiological function of the 67 kDa calcimedin (annexin VI). The annexins are a family of seven structurally related calcium- dependent phospholipid binding proteins. Our current results demonstrate that muscle tissues have the highest concentrations of annexin VI. Anti-annexin VI co-localizes with the calcium-pump, suggesting an association with the sarcoplasmic reticulum (SR). In addition, fractionation studies of SR demonstrate co-purifiiation of annexin VI with terminal cisternae. We propose four specific aims necessary to define the physiological function of annexin VI: (1) Binding constants for phospholipids and calcium will be determined in order to to evaluate the physiological significance of regulation; (2) Anti-annexin VI antibodies will be co-localized with the calcium-pump, calsequestrin and the ryanodine-sensitive calcium release channel; (3) Reconstitution studies of HSR proteins and fractions will be performed and in planar lipid bilayers in order to further resolve the regulatory role of annexin VI in calcium-dependent calcium release. Photoaffinity labeling studies will be employed to identify annexin VI target proteins in isolated terminal cisternae; and (4) The expression of annexin VI will be selectively depressed through the use of permeable, stable anti-sense RNA S-oligo nucleotides in cultured mouse muscle cells. The phenotypes examined will be rates of contraction/relaxation and dynamics of calcium release/uptake. Information on the function of annexin VI in skeletal muscle will be obtained by correlating localization studies in this highly ordered tissue with regulation of calcium release/uptake in a well-defined organelle, the SR, and with alteration in a highly differentiated phenotype, contraction. Our studies will provide definitive evidence for the physiological role of annexin VI in maintaining intracellular calcium homeostasis.

Calcium plays an essential role in the regulation of excitation-contraction coupling. Alterations in the regulation of intracellular levels of calcium is the molecular basis of many muscle diseases including muscular dystrophy and malignant hyperthermia. The objective of this proposal is to identify the cellular role of annexin VI in regulating the ryanodine-sensitive calcium-release channel found in the terminal cisternal of skeletal muscle sarcoplasmic reticulum. The annexins are a family of structurally related proteins which bind phospholipids in a calcium-dependent manner. Our current studies demonstrate annexin VI to be localized in skeletal muscle sarcoplasmic reticulum. We have also shown annexin VI to be a potent regulator of the reconstituted ryanodine-sensitive calcium-release channel. The proposed studies are designed to define the physiological function of annexin VI in skeletal muscle and in non-muscle cells. Differential extraction of cultured muscle cells will be used with antibodies against sarcoplasmic reticulum marker proteins (calsequestrin, calcium-release channel and calcium-pump) to resolve the cellular compartment of annexin VI. These studies will be complemented with immunogold EM. Reconstitution studies will be performed in planar lipid bilayers in order to identify regulatory proteins associated with annexin VI. These results will be complemented with photoaffinity labeling experiments designed to identify annexin VI binding proteins. The expression of annexin VI will be selectively suppressed with anti-sense RNA oligo nucleotides in muscle and non-muscle cells. We will the determine whether reduced annexin VI levels alters cellular function in vivo.

Calcium is a primary regulator of numerous functions in all cells. Changes in the levels of intracellular free calcium act as a signal. The mediation of intracellular free calcium is through high-affinity calcium binding-proteins. Calmodulin, a well-characterized calcium mediator protein, has been shown to be an essential gene product for cell viability. The Ca2+-calmodulin complex has been implicated in coupling cell responses to many stimuli. We have designed an approach to identify peptides which bind to a targeted protein, calmodulin. Ca2+-dependent affinity chromatography has been used to select calmodulin binding peptides from a bacteriophage library of random peptides. Sequence and predicted structure analysis of these peptides suggest that they are unique when compared to peptides previously reported as binding calmodulin. We propose to design an affinity-purification strategy to select sequences which are specific for the different conformational states of calmodulin, that is peptides which bind calmodulin only in the presence of calcium, those which bind only in the absence of calcium, and those which are indifferent to the calcium concentration. The physiological effects of these unique peptides will then be characterized in intact cellular systems including in the muscle fiber, neuron, chromaffin cell and epithelial cell. Synthetic genes will be designed which will be used for the expression of the calmodulin binding peptides in vivo. Cell growth, division and morphology will be examined as well as the stress response to elevated temperature and hypotonic challenge. Finally, selected calmodulin binding peptide sequences will be fused with promotor sequence in order to target expression of individual calmodulin binding peptides to the type II epithelial cells of the ling or cardiac ventricles of transgenic mice. These animals should allow for the understanding of calmodulin inhibition in intact tissue and the development of lung epithelial disease models such as cystic fibrosis and cardiac myopathies in male modifiers through selection from random peptide libraries is an independent approach for the study of cellular function. This peptide approach should be applicable to the evaluation of the role of other cellular proteins for which natural modifiers have yet to be discovered.

Calcium plays an essential role in the regulation of excitation-contraction coupling. Alterations in the regulation of intracellular levels of calcium is the molecular basis of many muscle diseases including muscular dystrophy and malignant hyperthermia. The objective of this proposal is to identify the cellular role of annexin VI in regulating the ryanodine-sensitive calcium-release channel found in the terminal cisternal of skeletal muscle sarcoplasmic reticulum. The annexins are a family of structurally related proteins which bind phospholipids in a calcium-dependent manner. Our current studies demonstrate annexin VI to be localized in skeletal muscle sarcoplasmic reticulum. We have also shown annexin VI to be a potent regulator of the reconstituted ryanodine-sensitive calcium-release channel. The proposed studies are designed to define the physiological function of annexin VI in skeletal muscle and in non-muscle cells. Differential extraction of cultured muscle cells will be used with antibodies against sarcoplasmic reticulum marker proteins (calsequestrin, calcium-release channel and calcium-pump) to resolve the cellular compartment of annexin VI. These studies will be complemented with immunogold EM. Reconstitution studies will be performed in planar lipid bilayers in order to identify regulatory proteins associated with annexin VI. These results will be complemented with photoaffinity labeling experiments designed to identify annexin VI binding proteins. The expression of annexin VI will be selectively suppressed with anti-sense RNA oligo nucleotides in muscle and non-muscle cells. We will the determine whether reduced annexin VI levels alters cellular function in vivo.

Antiphospholipid Syndrome (APS), also known as Hughes Syndrome, is a multiorgan vascular disease associated with myocardial infarction, stroke thrombosis and recurrent fetal loss. Forty- five percent of the people under the age of fifty who suffer strokes also demonstrate elevated levels of phospholipid antibody. Our hypothesis is that the presence of antiphospholipid antibodies in the circulation leads to vascular disorders, and that the escalating tissue damage can be abated with agents that bind the destructive antibodies. In order to test this hypothesis, it is necessary to create a genetically defined APS mouse that secretes antiphospholipid antibodies into the blood. We have produced and characterized a mouse monoclonal antibody which specifically recognizes phosphatidylserine. Preliminary data demonstrates that our monoclonal antibody binding specificity is similar to antibodies present in APS patients; this monoclonal antibody is therefore appropriate to develop a treatment model. The heavy and light chain of our monoclonal antiphospholipid antibody will be cloned by RT-PCR. These cDNAs will include the endogenous secretory peptide signal sequence. Bigenic mice will be used to direct ligand induced expression in the liver. Each monoclonal immunoglobulin chain will be differentially tagged with HA or FLAG epitopes. The transgenic animals will be characterized and compared with normal animals for the hallmark symptoms of APS, including prolonged in vitro coagulation times, recurrent fetal loss and vascular disease. An additional goal of this proposal is to develop treatment strategies for APS. Our approach is to neutralize the disease-causing antibodies. Peptides which bind and block the monoclonal antiphospholipid antibody in vitro can be selected from a combinatorial phage-display library of random peptides. Isolated peptides will be assessed for the ability to reduce the symptoms of APS displayed by this transgenic mouse. This study will produce a genetically-defined mouse model of APS which will prove useful for developing treatment strategies to ameliorate the symptoms associated with APS.