This proposal is concerned with the molecular and anatomical investigation of two families of neurotransmitter receptors - the dopamine receptor family and the serotonin receptor family. Both belong to the so-called """"""""seven transmembrane domains"""""""" superfamily which is characterized by receptors encoded in a single protein with seven putative alpha-helical stretches which span the cell membrane and form a pore to which the ligands bind. These receptors share a number of characteristics, including the fact that they transduce their signals by coupling to G proteins, and that they exhibit extensive sequence homologies, both within and across families. Our focus on the dopamine and the serotonin receptors is due to the role of these proteins in mediating the actions of two important monoaminergic systems which have been implicated in psychiatric disorders - i.e. dopamine and its hypothesized role in schizophrenia, and serotonin and its proposed role in depression. The proposal places the larger emphasis on the dopamine receptors, their structure, regulation and expression in limbic and motor systems, and secondary emphasis on serotonin receptors and their relationship to the stress axis. We propose to study all three known members of the dopamine receptor family - D1, D2, D3, at two levels: a) a molecular/structural level in which we examine the physical characteristics of these receptor proteins in an attempt to shed some light on their physic al configuration, describe the key features of their ligand binding pocket and tease out the molecular basis for their pharmacological selectivity. b) an anatomical/regulatory level, in which we complete the neuronal mapping of the mRNA's coding for these receptors, describe their relationship to each other and to dopaminergic and dopaminoceptive neurons, study the differential expression of unique receptor subtypes in neurons with particular biochemical identities (as indicated by the neurotransmitters and neuroregulators which they express), and examine the consequences of chronic agonist and antagonist treatment on the mRNA levels. These studies will complement and extend the existing body of knowledge based on receptor binding regarding the distribution and regulation of these receptors. We shall rely on in situ hybridization, receptor autoradiography and pharmacological tools for the anatomical/regulatory studies. For the structural studies, we shall use protein expression, domain swapping, site directed mutagenesis and molecular modeling. The serotonin receptor component of this proposal is concerned primarily with the expression and regulation of the multiple cloned members of this family in brain areas of relevance to the stress axis. Of particular interest is the apparent cross-regulation between these receptors and corticosteroid receptors, as well as their potential regulation by stress- related manipulations and by antidepressant drugs with high selectivity for serotonin. These experiments will employ the same anatomical, molecular and pharmacological tools described for the dopamine receptor studies. The findings of this project will be directly relevant to our analyses of post-mortem human brains proposed under Project III.
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