Studies will be undertaken to clone prospective alpha2-adrenergic receptor subtypes and to understand the molecular structure of alpha2-adrenergic receptors. At present the alpha2-adrenergic receptors can be classified into the alpha2-C10, alpha2-C4 and alpha2-C2 subtypes on the basis of molecular biological studies. On the basis of pharmacological studies, the alpha2-adrenergic receptors can be classified clearly into the alpha2A and alpha2B subtypes; additionally, there is suggestive evidence for two more subtypes. The alpha2-C10 and alpha2A subtypes are equivalent and recent data indicates that the alpha2-C2 and alpha2B subtypes are the same. The relationship of the alpha2-C4 to the pharmacologically defined subtypes is less clear, although, it may be equivalent to the alpha2B as characterized in brain tissue. Preliminary results clearly support the possibility of a fourth alpha2-adrenergic receptor subtype. The known DNA sequences of the alpha2-C10, alpha2-C4 and alpha2-C2 subtypes will be used to design oligonucleotide probes for use in the polymerase chain reaction (PCR). This powerful technique, which can be used to clone related genes, will be used in conjunction with traditional methods to identify new alpha2- receptor subtypes. Mutagenesis studies will be done to understand and define the ligand binding site of present and future members of alpha2-adrenergic receptor family. These studies will involve site-directed mutagenesis and PCR-based approaches. Recombinant DNA techniques will also be used to study palmitoylation of the alpha2-adrenergic receptor subtypes and to design receptor-fusion proteins that can be expressed in prokaryotic cells. These studies will help characterize important biochemical features of the alpha2-adrenergic receptor subtypes. The information that will be obtained by the proposed studies will help us understand the pharmacological characteristics of alpha2-adrenergic receptor subtypes. Drugs acting via alpha2-adrenergic receptors are presently used as sedatives and to treat hypertension. Additionally, alpha2-adrenergic agents have potential in the treatment of drug abuse, diabetes, memory loss and weight control. Some of these conditions represent serious public health problems that are difficult to treat and that may get worse as our nation deals with drug abuse and an aging population. The work being proposed here will provide a basis from which therapeutic solutions to these problems may be found.
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