The long term goal of this project is to understand the mechanisms by which adenosine receptors regulate ion transport. In ion transporting epithelia, adenosine plays a central role is a regulatory link between cellular energy demand and availability. Extracellular adenosine, released in proportion to cellular work, acts as an autacoid activator of specific seven transmembrane (7TM) G-protein coupled adenosine receptors. We have cloned, sequenced, and functionally characterized a new adenosine receptor regulating chloride transport (AoAR) appears to be the ancestral receptor from which mammalian adenosine receptors evolved. The AoAR has extraordinary functional and structural features that will be exploited in these studies including: (1) dual function (coupling to both stimulation and inhibition of chloride secretion); (2) unique N terminal, terminal and transmembrane residues and a unique intracellular loop involved in G-protein coupling; and (3) location of basic amino acids in certain domains permitting higher levels of protein expression of the receptor in E. coli than other G coupled receptors. By exploiting advantages of the rectal gland model system we will pursue the following specific aims: 1) We will identify the specific residues that are essential for the unique binding of agonists and antagonist to AoAR in a limited number of experiments. 2) We will challenge the paradigm that a hypothesis is that endogenous adenosine acts at AoAR to both inhibit and stimulate transepithelial Cl- secretion in the shark rectal gland. We will examine the coupling of AoAR to multiple G- proteins both in CHO cells expressing system, we have obtained highly purified AoAR with high specific binding activity. The structure of AoAR in having basic amino acids in all extracellular and intracellular loops confers the remarkable characteristic of Ao to be produced at high densities at high densities in inclusion bodies in E. coli. Using a 6 His purification scheme, coupled with further improvement in purifications (ion exchange and antagonist columns) and refolding steps, we will purify the active Ao adenosine receptor in sufficient amounts for structural studies of this protein.
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