Adrenergic receptor as a proto-oncogene

• US 5,573,908 A
• Filed: 12/20/1993
• Issued: 11/12/1996
• Est. Priority Date: 12/16/1991
• Status: Expired due to Term

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Allen, Lee F., Cotecchia, Susanna, Lefkowitz, Robert J., Caron, Marc G.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US08/170,684
• Time in Patent Office: 1,058 Days
• Field of Search: 435/7.2, 435/240.1, 435/6
• US Class Current: 435/6.14
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/9433 (Nor)adrenaline

Abstract

A recombinant cell comprising a host cell containing a recombinant DNA sequence is disclosed. The recombinant DNA sequence comprises vector DNA and DNA which encodes a mammalian adrenergic receptor. The host cell is one capable of undergoing proliferation in response to activation of the adrenergic receptor. In one specific embodiment of the foregoing, the adrenergic receptor includes a mutation in the third cytoplasmic loop thereof which renders the adrenergic receptor constitutively active, and the host cell undergoes proliferation in response to the constitutively active adrenergic receptor. Also disclosed are in vitro assays employing the foregoing which are useful for screening test compounds for antitumor and antiatherogenic activity, along with a diagnostic assay for detecting the oncogenic activation of cells in a patient. The diagnostic assay comprises collecting sample cells which express adrenergic receptors from the patient, and then detecting the presence or absence of a mutation in the adrenergic receptor which renders the receptor constitutively active. The presence of such a mutation indicates the oncogenic activation of the cells.

Claims

1. An in vitro assay procedure, comprising:

contacting a test compound to a recombinant cell, the recombinant cell comprising a mammalian host cell containing a heterologous DNA molecule, the heterologous DNA molecule comprising vector DNA and DNA which encodes a mammalian α

₁ -adrenergic receptor, wherein said mammalian α

₁ -adrenergic receptor is expressed in said host cell, and wherein said recombinant cell undergoes proliferation in response to activation of said mammalian α

₁ -adrenergic receptor; and

detecting whether or not said test compound inhibits the proliferation of said recombinant cell.

2. An assay method according to claim 1, further comprising the step of contacting an adrenergic receptor agonist to said recombinant cell in an amount effective to activate proliferation of said recombinant cell, and wherein said detecting step is carried out by detecting whether said test compound inhibits the proliferation of said recombinant cell caused by said adrenergic receptor agonist.

3. An assay method according to claim 1, wherein said mammalian α

₁ -adrenergic receptor includes a mutation in the third cytoplasmic loop thereof rendering said receptor constitutively active, wherein said recombinant cell undergoes proliferation in response to said constitutively active adrenergic receptor, and wherein said detecting step is carried out by detecting whether said test compound inhibits the proliferation of said recombinant cell caused by said constitutively active mammalian α

₁ -adrenergic receptor.

4. A method according to claim 1, wherein said host cell is a fibroblast cell.

5. A method according to claim 1, wherein said recombinant cell is grown as a monolayer in in vitro culture.

6. An assay method according to claim 1, wherein said mammalian α

₁ -adrenergic receptor includes a mutation in the third cytoplasmic loop thereof rendering said receptor constitutively active, wherein said recombinant cell undergoes proliferation in response to said constitutively active adrenergic receptor, and wherein said detecting step is carried out by detecting whether said test compound inhibits the proliferation of said recombinant cell caused by said constitutively active mammalian α

₁ -adrenergic receptor.

7. An in vitro assay procedure, comprising:

contacting a test compound to a recombinant cell grown as a monolayer in in vitro culture, the recombinant cell comprising a mammalian fibroblast host cell containing a heterologous DNA molecule, the heterologous DNA molecule comprising vector DNA and DNA which encodes a mammalian α

₁ -adrenergic receptor, wherein said mammalian α

₁ -adrenergic receptor is expressed in said host cell, and wherein said recombinant cell undergoes proliferation in response to activation of said mammalian α

₁ -adrenergic receptor; and

detecting whether or not said test compound inhibits the proliferation of said recombinant cell.

8. An assay method according to claim 7, further comprising the step of contacting an adrenergic receptor agonist to said recombinant cell in an amount effective to activate proliferation of said recombinant cell, and wherein said detecting step is carried out by detecting whether said test compound inhibits the proliferation of said recombinant cell caused by said adrenergic receptor agonist.