Edward Herbert, PhD - US grants

In the past two or three years it has been shown that there are at least three different genes involved in the production of opioids. One gene codes for pro-opiomelanocortin (POMC) from which Beta-endorphin, ACTH and melanocyte stimulating hormones are derived. A second gene codes for pro-enkephalin which contains multiple copies of leu-and met-enkephalin and other enkephalin peptides. A third gene codes for prodynorphin, the common precursor to Alpha-neo-endorphin and dynorphin. The major long range goal of the proposed research is to better understand how the expression of each of the opioid peptide genes is regulated in the pituitary, brain and other sites in the body. We will use cDNA probes isolated from cDNA clone banks to identify and sequence the genes and determine their chromosomal locations. Specific genomic DNA probes will then be used to study how expression of the genes is regulated at the levels of transcription, and metabolism of the mRNA using intact animals, cell lines, and primary cell culture systems. Genomic DNA probes isolated from lambda genomic libraries will be made to specific regions of each gene to determine if the genes undergo any structural change (i.e. methylation) or rearrangements following teatment with various agents, or during the onset of pathological processes that affect expression of the genes. These molecular hybridization probes will also be used to determine where each class of peptide is synthesized in laboratory animals and humans by measuring mRNA levels in various tissues. In addition, we will determine the effect of various regulators and drugs such as morphine and steriods on the mRNA levels in each tissue and in different neuronal pathways. Finally, transfer of genes and segments of genes from expressing cell types to non-expressing cell types (and vice versa) will be performed to delineate those portions of the genes and their flanking sequences that are required for expression. Gene transfer experiments will also be performed to define specific amino acid sequences in the precursor proteins that are necessary for correct proteolytic processing of these polyproteins.

gene expression; melanocyte stimulating hormone; corticotropin releasing factor; nucleic acid sequence; neuroendocrine system; genetic manipulation; glucocorticoids; hormone regulation /control mechanism; genetic transcription; genetic translation; neural information processing; messenger RNA; tissue /cell culture; brain mapping; flow cytometry; molecular cloning; human tissue; complementary DNA; endonuclease; ultracentrifugation; gel electrophoresis; radiotracer; autoradiography;

Opioid peptides are found in many different sites in the nervous and endocrine systems where they mediate diverse types of responses. All numbers of this large family of peptides (more than 15) have been shown to be derived from 3 different precursors known as pro-opiomelanocortin, proenkephalin and prodynorphin. The receptors that opioid peptides bind to are also diverse in nature. They can be classified into 3 predominant types based on the differences in opioids they bind. Each type of receptor and opioid peptide has a somewhat different distribution in the brain and may mediate different kinds of responses. The major goal of this research is to further our understanding of the mechanisms that regulate expression of opioid peptides and opioid receptors. This goal will be accomplished by a multilevel approach employing methods of molecular biology, immunology and protein chemistry. The sites of synthesis of opioid peptides will be mapped in the brain by in situ hybridization techniques using DNA probes specific for each opioid precursor. The regulatory sites in the opioid genes will be defined by gene transfer techniques. This will be done by studing the effects of specific alterations of the opioid genes (site-specific in vitro mutagenesis) on transcriptional activity and on processing of the precursors in two different types of recipient cells (mammalian cell lines and frog oocytes). The first step in the study of opioid receptors will be to determine the structure of one type of receptor by recombinant DNA techniques. A relatively new approach called the hybridization selection procedure will be used to purify the mRNA species that code for the receptor. The frog oocyte expression system will be used to assay the mRNA for its capacity to direct the synthesis of the receptor proteins. cDNA will be prepared from the purified mRNA and cloned. From the sequence of the cDNA it will be possible to determine the number of subunits in the receptor and the structure of each subunit. The next step will be to use various receptor cDNA probes to determine the number of opioid receptor genes and how similar these genes are to one another.

Neuropeptides are synthesized as large precursor proteins which undergo specific proteolytic cleavages and modifications to produce bioactive peptides. The initial cleavages are thought to occur at pairs or basic amino acid residues to produce peptides with C-terminal Lys of Arg residues. The Lys or Arg residues are removed by carboxypeptidase-like enzymes to produce bioactive peptides. Although the proteolytic reactions are critical steps in activating neuropeptides, we know very little about the endopeptidases and exopeptidase that catalyze these reactions. Recently, a member of the kallikrein family of serine proteases has been implicated in the endoproteolytic cleavage of pro-opiomelanocortin (POMC) in the pituitary. By use of a mouse kallikrein cDNA probe we have been able to demonstrate that a single kallikrein protease is present in the mouse pituitary tumor cell line that produces POMC. A carboxypeptidase enzyme has been implicated in proenkephalin processing in the bovine adrenal medulla. This enzyme has been purified and partially sequenced. Several approaches will be used to determine whether the kallikrein and carboxypeptidase enzymes are required for POMC and proenkephalin processing. We will first determine the effect of selectively removing these proteins from the cellular protein pool on processing of the two precursors. This will be done by a gene transfer technique. Second, the subcellular and cellular distribution of the enzyme will be determined. Third, we will test the ability of specific inhibitors of these enzymes to alter processing of the precursors. Fourth, we plan to determine the structure of the genes that code for these enzymes. Control of expression of these genes at the transcriptional level will also be analyzed to determine whether activity of these genes is regulated coordinately with the activity of precursor genes. A final objective will be to determine when the enzyme genes are turned on relative to the precursor genes during embryonic development.