CLONING AND MUTAGENESIS OF A2 - ADRENERGIC RECEPTORS

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.