The proposed molecular neurobiological studies seek to provide information leading to a better understanding of the effects of morphine and other drugs of abuse on human physiology. The objectives of this Scientist Development Award (K21) proposal are to determine the effects of drugs of abuse and hormones on the expression of the human mu-opioid receptor gene and to study the mechanisms that regulate expression by identifying the DNA sequences and protein factors involved. Substances which alter mRNA level may act on mRNA stability; however, our proposal focuses on regulation of transcription. A reporter gene construct is engineered using the promoter of the human mu-opioid receptor which has been cloned by the candidate. This promoter is ligated to an easily assayed reporter gene. Transfer of the reporter construct into cell cultures and transgenic mice is used to analyze the promoter function. Opioid receptor expressing and non-expressing cell lines are compared to determine the genetic basis for tissue specificity of mu-opioid receptor expression Gene transfer of the reporter construct into transgenic mice provides an in vivo test for promoter regions that confer tissue specificity. If the reporter protein is produced in a manner characteristic of mu-opioid receptor expression, then the transgenic mice and the cells will serve as sensitive and quantitative models for testing mu-opioid expression in response to a variety of experimental protocols. The reporter constructs are also used to study mu-opioid receptor expression during development in both cell culture and transgenic mouse models. Cell cultures are studied prior to, during, and following differentiation into a mature, neuron-like phenotype. Transgenic mice are studied during embryogenesis. The effects of chronic, acute, and subacute treatment with agonists (e.g. morphine or opioid peptides) on opioid receptor rRNA level and transcription rate are tested. Treatments that alter transcription rate are examined in cell cultures transfected with the reporter constructs. Transcription regulation in response to treatment with other drugs of abuse (e.g. cocaine, or ethanol), steroid hormones, or interleukins is also examined. Constructs bearing deletions or mutations in the promoter regulatory regions will have unregulated expression of the reporter. DNA:protein binding assays (e.g. electrophoresis mobility shift assays and DNA footprinting) are used to further characterize regions of the promoter DNA involved in regulating transcription. The protein transcription factors that bind these regions can also be characterized. Any novel transcription factors that regulate mu-receptor transcription may be cloned so that they can be more easily and more fully characterized. Once regulatory regions of the promoter have been found, then reporter constructs having mutations in the regulatory sequences are transfered into mice. If these transgenic mice also express the reporter protein independent of regulation, then constructs will be prepared in which the reporter gene is replaced with the mu-opioid receptor coding sequence. Transgenic mice bearing such constructs may produce mu-opioid receptors independent of normal regulation. These mice may be resistant to developing tolerance and dependence, although desensitization through post-translational modification would be expected to remain unaffected.
| Andria, M L; Simon, E J (1999) Localization of promoter elements in the human mu-opioid receptor gene and regulation by DNA methylation. Brain Res Mol Brain Res 70:54-65 |
