The United States of America as Represented by the Secretary of

The United States of America as Represented by the Department of Health

The United States of America

Lawrence C Mahan

David Robert Sibley

Frederick J Monsma, Jr

Loris D McVittie

Michael Jay Brownstein

J Stephen Fink

Jon Marsh

Monika Konig

The present invention relates, in general, to a method of identifying ligands and antagonists of ligands. In particular, the present invention relates to a method of identifying ligands and antagonists of ligands which bind to cloned G.sub.8 - or G.sub.1 -coupled receptors. The present invention also relates to a cell that comprises a recombinant cyclic AMP sensitive reporter construct.

Method of identifying ligands and anatgonists of G-protein coupled

The present invention relates to the molecular cloning and expression of the D.sub.1 dopamine receptor protein that is linked to the activation of adenylyl cyclase activity. By constructing cell lines that express the D.sub.1 receptor, the affinities and efficacies of agonist and antagonist drugs with the receptor can be assessed. The present invention further relates to a recombinant DNA construct that includes a vector and a DNA fragment encoding the D.sub.1 receptor. The present invention also relates to a host cell transformed with a recombinant DNA construct, so that the DNA fragment is expressed and the D.sub.1 receptor is produced. Suitable expression systems include eukaryotic and procaryotic cells, especially mammalian cells such as rat or human. The present invention further relates to the antibody to the D.sub.1 receptor protein. For diagnostic purposes, antibodies to this receptor can be prepared by producing all or a portion of the receptor protein and injecting these into various types of mammals. Using the resulting antibodies, expression of the D.sub.1 receptor cDNA in cells can be measured.

cDNA encoding a rat D.sub.1 dopamine receptor linked to adenylyl cyclase

In the present investigation, we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D.sub.2 receptor CDNA.sup.12. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the 3rd cytoplasmic loop, a region believed to be involved with G protein coupling. This is the first example of a novel G-protein coupled receptor isoform generated by alternative RNA splicing.

cDNA encoding the long isoform of the D.sub.2 dopamine receptor

In the present investigation, we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D.sub.2 receptor cDNA.sup.12. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the 3rd cytoplasmic loop, a region believed to be involved with G protein coupling. This is the first example of a novel G-protein coupled receptor isoform generated by alternative RNA splicing.

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Bethesda, MD, United States of America

Lawrence C Mahan