The long-term objective of our research is to elucidate the molecular mechanisms that cause the progressive blinding disease retinitis pigmentosa (RP). Recent work showed that inappropriate transducin signaling activity caused degeneration in a subset of RP mutations. Genetic ablation of transducin function protected from degeneration. However, another subset of mutants degenerated in a transducin-independent manner and showed partial protection when dark-reared. This suggests that inappropriate rhodopsin mediated activation of a G-protein independent pathway may be a cause of RP. Over the past decade, compelling evidence has accumulated to suggest that in addition to traditional G-protein signaling pathways, activated 7- transmembrane (7-TM) receptors can activate G-protein independent pathways. For a few receptors, receptor/arrestin complexes mediate cell signaling. MAP kinase and tyrosine kinase signal transduction pathways have been implicated in these alternative pathways. In this R21 application, we propose to test the hypothesis that light-activated rhodopsin or rhodopsin/arrestin complexes activate G-protein independent signaling pathways. We will isolate alternative pathway(s) using transgenic mice with null mutations in the transducin a-subunit (Tra-/-) and/or arrestin (Arr-/-) genes.
Specific Aim 1 will test the prediction that rhodopsin/arrestin signaling complexes form in a light-dependent manner in Tra-/- rods. We will look for association of arrestin with membrane fractions after light exposure by Western blot analysis.
Specific Aim 2 will test the prediction that in the absence of functional transducin, light- activated rhodopsin activates MAP kinase and/or tyrosine kinase signal transduction pathways. We have targeted for investigation 17 kinase-signaling molecules that are enriched in photoreceptor cells. A hallmark feature of signal activation is protein phosphorylation. To identify G-protein independent signaling pathways, we will compare the phosphorylation state of signal kinases in dark-adapted and light exposed rod outer segment preparations from wild-type, Tra-/- and Tra-/-, Arr-/- double mutant mice. We expect activation only in light-exposed animals. The proposed studies will provide a basic understanding of G- protein independent signaling by 7-TM receptors. This provides the basis for investigation of non-traditional signal transduction pathways in human retinal degenerations and other 7-TM receptor diseases. ? ? ?
