The interaction of the bovine opsin apoprotein with transducin in rod outer segment membranes was investigated using a guanyl nucleotide exchange assay. In exhaustive binding experiments, opsin activates transducin, with half-maximal exchange activity occurring at 0.8 mol of opsin/mol of transducin. The opsin activity was light-insensitive, hydroxylamine-resistant, unaffected by stoichiometric concentrations of retinaloxime, and more heat-labile than rhodopsin. The t1/2 of transducin activation in the presence of excess opsin was 8.5 min, compared with 0.7 min for metarhodopsin (II). The second-order rate constants were determined to be 0.012 pmol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) bound per min/nM opsin and 0.35 pmol of GTP gamma S bound per min/nM metarhodopsin (II). Opsin was able to activate more than one transducin, although there appeared to be a turnover-dependent inactivation of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for optimal activity, with no activity in buffers of pH > 9, whereas metarhodopsin (II) exhibited activity at pH > 9. Regulation of opsin activity by stoichiometric amounts of retinal was observed, with inhibition by 11-cis-retinal and stimulation by all-trans-retinal. A model for opsin activity is proposed.

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The pineal gland expresses a unique member of the opsin family (P-opsin; Max, M., McKinnon, P. J., Seidenman, K. J., Barrett, R. K., Applebury, M. L., Takahashi, J. S., and Margolskee, R. F. (1995) Science 267, 1502-1506) that may play a role in circadian entrainment and photo-regulation of melatonin synthesis. To study the function of this protein, an epitope-tagged P-opsin was stably expressed in an embryonic chicken pineal cell line. When incubated with 11-cis-retinal, a light-sensitive pigment was formed with a max at 462 ؎ 2 nm. P-opsin bleached slowly in the dark (t 1/2 ‫؍‬ 2 h) in the presence of 50 mM hydroxylamine. Purified P-opsin in dodecyl maltoside activated rod transducin in a light-dependent manner, catalyzing the exchange of more than 300 mol of GTP␥S (guanosine 5-O-(3-thiotriphosphate))/mol of P-opsin. The initial rate for activation (75 mol of GTP␥S bound/mol of P-opsin/min at 7 M) increased with increasing concentrations of transducin. The addition of egg phosphatidylcholine to P-opsin had little effect on the activation kinetics; however, the intrinsic rate of decay in the absence of transducin was accelerated. These results demonstrate that P-opsin is an efficient catalyst for activation of rod transducin and suggest that the pineal gland may contain a rodlike phototransduction cascade.

ABSTRACT:The aberrant activation of B-cells has been implicated in several types of cancers and hematological disorders. BTK and PI3Kδ are kinases responsible for B-cell signal transduction, and inhibitors of these enzymes have demonstrated clinical benefit in certain types of lymphoma. Simultaneous inhibition of these pathways could result in more robust responses or overcome resistance as observed in single agent use. We report a series of novel compounds that have low nanomolar potency against both BTK and PI3Kδ as well as acceptable PK properties that could be useful in the development of treatments against B-cell related diseases.