Gene encoding the rat dopamine D4 receptor

Abstract

A gene, flanking 5′ and 3′ sequences and derived cDNA encoding a rat D 4 dopamine receptor that is predominantly located in the cardiovascular and retinal systems is disclosed. The cDNA has been expressed in transfected mammalian cells and demonstrated to preferentially bind dopamine antagonists such as clozapine. The cDNA is useful as a probe for related D 4 dopamine receptors. Expressed in appropriate cell lines, it is useful as an in vitro screen for drugs which specifically bind to the receptor. Drugs that specifically bind to the receptor are then screened using standard methodology in rats, mice or dogs, for the physiological effects. Amino acids deduced from the determination of cDNA can be used to generate either polyclonal or monoclonal antibodies which recognize the D 4 receptor sequence but do not recognize D 1 , A 2 , D 3 or D 5 donpamenergic receptors, for use in immunocytochemical studies, identification and isolation via flow sorting of D4 expressing cell types. Antibodies could also be used to block or modify the effects of D4 agonists and/or antagonists. It is also demonstrated that selective stimulation or inhibition of some dopamine receptors, including D 4 , can be used to induce changes in the morphology of cells such as neurons.

We claim:  
     
         1 . An isolated nucleic acid molecule encoding a rat D 4  dopamine receptor.  
     
     
         2 . The molecule of  claim 1  further comprising the flanking 5′ and 3′ untranalated sequences.  
     
     
         3 . The molecule of  claim 1  wherein the molecule includes the untranslated introns of the derived gene.  
     
     
         4 . The molecule of  claim 1  consisting essentially of sequence ID No. 1.  
     
     
         5 . The molecule of  claim 1  encoding the protein consisting essentially of sequence ID No. 2.  
     
     
         6 . The molecule of  claim 1  in an expression cell system.  
     
     
         7 . The molecule of  claim 6  in a non-rat D4 expressing cell.  
     
     
         8 . A probe for dopamine receptors comprising at least fourteen contiguous nucleotides of a nucleic acid molecule encoding a rat D 4  dopamine receptor as shown in Sequence ID No. 1.  
     
     
         9 . The probe of  claim 8  further comprising a label selected from the group consisting of dyes, radiolabels, tomography positron emission labels, magnetic resonance imaging labels, fluorescent labels, and enzymes.  
     
     
         10 . A method for screening for compounds selectively binding to a rat D 4  dopamine receptor comprising 
 transfecting cells with an isolated nucleic acid molecule encoding a rat D 4  dopamine receptor,    exposing the cells expressing the dopamine receptor to compounds which may bind to the receptor,    selecting those compounds binding to the receptor,    comparing the binding of the selected compounds with the binding of the compounds to other dopamine receptors, and    determining which compounds bind to the D 4  dopamfine receptor but with lower affinity to other dopamine receptors.    
     
     
         11 . The method of  claim 10  further comprising administering the compounds to a biological material selected from the group consisting of animals, tissue explants, and individual cells and determining the effect on the physiology of the material.  
     
     
         12 . A method for screening for compounds selectively acting on tissue selected from the group consisting of the cardiovascular and retinal systems by binding to dopamine receptors comprising 
 transfecting cells with a nucleic acid molecule encoding a D 4  dopamine receptor, and    determining if compounds bind to the receptor and not to other dopamine receptors with the same affinity,    administering the compounds with the desired binding affinity to a biological material selected from the group consisting of animals, tissue explants, and individual cells and measuring effects on cardiovasculature and retinal physiology.    
     
     
         13 . The method of  claim 12  wherein the D 4  dopamine receptor is selected from the group consisting of human and rat D 4  dopamine receptors.  
     
     
         14 . The method of  claim 12  wherein the other dopamine receptors are selected from the group consisting of human and rat D 2  and D 3  receptors.  
     
     
         15 . A method for altering the morphology of mammalian cells comprising stimulating or inhibiting a dopamine receptor selected from the group consisting of D 2 , D 3 , and D 4  receptors.  
     
     
         16 . The method of  claim 15  wherein the cells do not normally express the dopamine receptor or wherein the cells do not normally express as many molecules of the dopamine receptor.  
     
     
         17 . The method of  claim 15  wherein the cells are neuronal cells.  
     
     
         18 . The method of  claim 15  wherein the dopamine receptors are selectively stimulated by exposure to an effective amount of a compound selected from the group consisting of dopamine antagonists and dopamine agonists.  
     
     
         19 . The method of  claim 15  wherein the expression of the receptors is selected to alter the morphology of the cells.  
     
     
         20 . The method of  claim 17  wherein the cells are stimulated by a compound binding to the dopamine receptors so that the neurites increase the number and extension of the neurites.  
     
     
         21 . The method of  claim 19  wherein the cells are stimulated by exposure to a compound selected from the group consisting of dopamine agonists, dopamine antagonists, antibodies to D 4 , and combinations thereof.  
     
     
         22 . An antibody immunoreactive with the rat D 4  receptor.  
     
     
         23 . The antibody of  claim 21  selected from the group consisting of polyclonal and monoclonal antibodies which recognize the D 4  receptor sequence but do not recognize D 1 , D 2 , D 3  and D 5  dopamenergic receptors.  
     
     
         24 . A method for treating disorders of the cardiovasoulature or retinal tissue comprising administering a compound selectively binding to D 4  receptors to a patient in need of treatment in an amount effect to decrease the symptoms of the disorder.  
     
     
         25 . The method of  claim 23  wherein the compound modulates the degree of vasoconstriction.  
     
     
         26 . A method for treating disorders of neuronal morphology or connections comprising administering a compound selectively binding to D 2 , D 3 , or D 4  receptors to a patient in need of treatment thereof in an amount effective to decrease the symptoms of the disorder.  
     
     
         27 . A method for preventing the development of disorders of neuronal morphology or connections comprising administering a compound selectively binding to D 2 , D 3 , or D 4  receptors to a patient at risk thereof in an amount effective to prevent or delay the onset of evidence of the disorder.  
     
     
         28 . The method of  claim 27  wherein the compound is selected from the group of compounds modulating the number, branching, or outgrowth or neurites.