The catecholamine dopamine plays a major role in the regulation of cognitive, emotional, and behavioral functions. Abnormalities in its regulation have been implicated in a number of psychiatric and neurological disorders. Dopamine exerts its actions at several G protein-coupled receptors, including the D2-like (D2, D3, D4) and D1-like (D1, D5) receptors. Most antipsychotic medications potently inhibit the D2, D3, and D4 receptors, and it is not clear which of these are relevant to the etiology and treatment of psychosis. Furthermore, the structural bases for the pharmacological differences among the D2-like receptors are unknown. For example, the atypical antipsychotic clozapine interacts with the D4 receptor with an affinity an order of magnitude higher than with the D2 and D3 receptors. The identified residues that affect agonist or antagonist binding to the D2 receptor, however, are completely conserved in all catecholamine receptors and cannot account, therefore, for such differences in binding specificities among these receptors. Additional residues must contribute to binding, either directly or indirectly, and the differences can only be explored by comparing a detailed mapping of the binding site in the different receptors. We have developed an approach, the substituted-cysteine accessibility method, to obtain information about the structure of binding sites by systematically identifying the residues which line the site. Our approach combines site-directed mutagenesis to substitute cysteine for putative membrane-spanning segment residues, heterologous expression of the mutant, and probes of the aqueous surface accessibility of the cysteine residue which utilize its ability to react with small, highly polar, charged, sulfhydryl-specific reagents. We have used this method to map the third, fifth, sixth, and seventh membrane-spanning segments of the D2 receptor and to determine activation-induced conformational changes in the homologous beta2 adrenergic receptor. The long-term goals of this project are (a) to understand the structural bases of agonist and antagonist binding and specificity in the dopamine D2-like receptors and related biogenic amine receptors and (b) to determine how agonist binding is transduced into G protein activation. Thus, I propose the following specific aims: (1) complete the identification of all the amino acid residues in membrane-spanning segments forming the surface of the binding site crevice of the dopamine D2 receptor, (2) examine the roles of the membrane-spanning residues in agonist potency and efficacy, (3) identify the residues which are responsible for differences in pharmacological specificity between the dopamine D2 and dopamine D4 receptors, and (4) identify conformational changes of the membrane-spanning segments concomitant with changes in the functional state of the receptor.