The overall objective of our research is to explain the biochemical mechanism of alpha2-adrenergic signal transaction. The immediate objective of our proposed research is to elucidate the structure, regulation and function of alpha2-adrenergic receptor with greater precision. To achieve this objective we hope to: 1. complete our biochemical and physicochemical characterization of the alpha2-adrenergic receptor protein. We will determine its: a) molecular weight by sedimentation equilibrium; b) partial amino acid sequence; c) helical conformation by circular dichroism; d) isoelectric point by isoelectric focusing; e) ability to autophosphorylate or act as a substrate for cyclic AMP- and cyclic GMP-dependent or cyclic nucleotide-independent protein kinases; 2. facilitate our biochemical and genetic studies on the alpha2-adrenergic receptor characterization and its regulation by producing polyclonal and nonoclonal antibodies against the purified receptor protein. This will enable us to develop a radioimmunossay (involving polyclonal antibodies) or immunoradiometric assay (involving monoclonal antibodies) to quantify the receptor; 3. biochemically characterize and determine the structure of the alpha2-agonist binding domain of the receptor protein; 4. biochemically determine if the alpha receptors in other mammalian tissues such as blood vessels, blood platelets, heart, liver, salivary glands, brain, kidney, and uterus are similar. This will initially involve utilization of the immunologic probes, polyclonal and monoclonal anti-bodies, structural comparison by peptide mapping and ultimately by the genetic probes. To determine the structure of the receptor proteim, we will complement the above biochemical and immunologic studies with the following genetic studies; 5. Complete our ongoing studies on molecular cloning of the alpha2- receptor gene; 6. Determine the nucleotide sequence of the cloned gene from which the amino acid sequence of the receptor will be inferred. The approaches proposed to achieve these aims involve complementary biochemical, immunologic, andrecombinant techniques.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Physiological Chemistry Study Section (PC)
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University of Tennessee Health Science Center
Schools of Medicine
United States
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Venkataraman, V; Duda, T; Sharma, R K (1999) alpha2D/A-adrenergic receptor gene induction in the retina by phorbol ester: involvement of an AP-2 element. Genes Cells 4:161-73
Duda, T; Venkataraman, V; Goraczniak, R et al. (1999) Functional consequences of a rod outer segment membrane guanylate cyclase (ROS-GC1) gene mutation linked with Leber's congenital amaurosis. Biochemistry 38:509-15
Duda, T; Venkataraman, V; Krishnan, A et al. (1998) Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) gene: structure, organization and regulation by phorbol ester, a protein kinase C activator. Mol Cell Biochem 189:63-70
Duda, T; Goraczniak, R M; Pozdnyakov, N et al. (1998) Differential activation of rod outer segment membrane guanylate cyclases, ROS-GC1 and ROS-GC2, by CD-GCAP and identification of the signaling domain. Biochem Biophys Res Commun 242:118-22
Krishnan, A; Goraczniak, R M; Duda, T et al. (1998) Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism. Mol Cell Biochem 178:251-9
Venkataraman, V; Duda, T; Sharma, R K (1998) The alpha(2D/A)-adrenergic receptor-linked membrane guanylate cyclase: a new signal transduction system in the pineal gland. FEBS Lett 427:69-73
Venkataraman, V; Duda, T; Sharma, R K (1997) The bovine alpha 2D-adrenergic receptor gene: structure, expression in retina, and pharmacological characterization of the encoded receptor. Mol Cell Biochem 177:113-23
Goraczniak, R; Duda, T; Sharma, R K (1997) Structural and functional characterization of a second subfamily member of the calcium-modulated bovine rod outer segment membrane guanylate cyclase, ROS-GC2. Biochem Biophys Res Commun 234:666-70
Sharma, R K; Duda, T; Goraczniak, R et al. (1997) Membrane guanylate cyclase signal transduction system. Indian J Biochem Biophys 34:40-9
Sharma, R K; Duda, T (1997) Plasma membrane guanylate cyclase. A multimodule transduction system. Adv Exp Med Biol 407:271-9

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