This project will study ciliary process (cp) proteins that are dephosphorylated by phosphatase enzymes specifically activated by signals generated when hormones or drugs bind to receptors. The goal is to establish mechanisms for biochemical regulation of aqueous humor secretion and to apply the knowledge to improved therapies for glaucoma. The immediate objectives are: a) to characterize five cp phosphoproteins of unknown structure and function that are regulated by phosphatase enzymes (PP-ase). The progression of studies to be done on each protein are: (i) separation from other phosphoproteins, (ii) analysis of phosphoamino acids, (iii) reconstitution with specific PP-ase activities, (iv) analysis of amino acids dephosphorylated by specific PP-ase activities, (v) scaleup of isolation/purification to get 1-5 mug protein, (vi) sequencing of 10-15 mer constituent peptides, (vii) synthesis of same peptides, preparation of antibody, confirmation that Ab recognizes phosphoprotein, (viii) synthesis of corresponding nucleotide probes, screening of a cp cDNA library with Ab and nucleotide probes, (ix) sequencing of cDNA in (+) clones, (x) confirmation of coded protein by in vitro translation. b) To characterize DARPP-32 functions and five DARPP-related proteins expressed in cp. For DARPP steps (v)-(vii) are not needed because the sequence is known and Ab's are available, but the other listed studies above will proceed for each of the DARPP-related proteins. c) To characterize ciliary process phosphoprotein phosphatases.
The aims of these enzyme studies are: (i) To separate different PP-ase activities in cp and characterize with specific regulator, modulator, inhibitor and substrate proteins, (ii) reconstitute separate activities with the 5 phosphoproteins isolated from cp, (iii) confirm that cp calcineurin specifically dephosphorylates DARPP-related phosphoproteins, (iv) characterize a novel cAMP-dependent PP-ase in cp, which has not been described elsewhere. The DARPP sequence and those of other phosphopeptides regulated by specific kinase/phosphatase activities could be of great significance. They may components of, or be incorporated into more complex functional proteins, such as ion channels or exchangers, thereby conferring on them a regulatory domain.
