Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110 -3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the ␤-subunit of the insulin receptor (IR␤) in ROS membranes, which leads to the association of PI3K enzyme activity with IR␤. Retinas from light-or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IR␤, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P 2 as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IR␤ and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IR␤ immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IR␤ in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P 3 . We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stressinduced apoptosis.Many cell proliferation and cell survival pathways are initiated upon activation of tyrosine kinase receptors, which transduce their signals by recruiting a variety of cytoplasmic signaling proteins (1, 2). Many of the signaling proteins contain phosphotyrosine binding domains, Src homology 2 (SH2) 1 domains, and Src homology 3 (SH3) domains, which are involved in mediating protein-protein interactions (4, 5).The phosphotyrosine-dependent interaction between different phosphotyrosine binding and SH2 domain-containing proteins with activated receptors initiates cellular changes that take place in response to a wide range of extracellular signals (2).One of the SH2 domain-containing proteins, phosphoinositide 3-kinase (PI3K), consists of an ϳ85-kDa regulatory subunit (p85) and a ϳ110-kDa catalytic subunit (p110), the latter being responsible for the phosphorylation of phosphoinositides at the D3 position and of serine residues in proteins (7, 8). The p85 subunit contains an SH3 domain capable of binding to proline-rich sequences, a p110 binding domain, and two SH2 domains. PI3K was initially found to be associated with middle-T/pp60c-Src (9), pp60v-Src, and platelet-derived growth factor receptors in both normal and transformed NIH3T3 fibroblast cells (10, 11). PI3K activity increases in response to platelet-derived growth factor binding to its receptor, in large part because the p85-p110 complex is translocated from the cytosol to the plasma membrane, by the direct binding of the p85 SH2 domain to tyro...

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, but the role of IR activity in vivo is unknown. We previously reported that light causes increased tyrosine phosphorylation of the IR in vivo, which leads to the downstream activation of the phosphoinositide 3-kinase and Akt pathway in rod photoreceptor cells. The functional role of IR in rod photoreceptor cells is not known. We observed that light stress induced tyrosine phosphorylation of the IR in rod photoreceptor cells, and we hypothesized that IR activation is neuroprotective. To determine whether IR has a neuroprotective role on rod photoreceptor cells, we used the Cre/lox system to specifically inactivate the IR gene in rod photoreceptors. Rodspecific IR knock-out mice have reduced the phosphoinositide 3-kinase and Akt survival signal in rod photoreceptors. The resultant mice exhibited no detectable phenotype when they were raised in dim cyclic light. However, reduced IR expression in rod photoreceptors significantly decreased retinal function and caused the loss of photoreceptors in mice exposed to bright light stress. These results indicate that reduced expression of IR in rod photoreceptor cells increases their susceptibility to lightinduced photoreceptor degeneration. These data suggest that the IR pathway is important for photoreceptor survival and that activation of the IR may be an essential element of photoreceptor neuroprotection. Insulin receptor (IR)2 signaling provides a trophic signal for transformed retinal neurons in culture (1), but the role of the IR in vivo is unknown. IR activation has been shown to rescue retinal neurons from apoptosis through a phosphoinositide 3-kinase (PI3K) cascade (1). We previously reported that light induces tyrosine phosphorylation of the retinal IR and that this activation leads to the binding of PI3K to rod outer segment (ROS) membranes (2). More recently, we demonstrated that IR activation is mediated through the G-protein-coupled receptor rhodopsin (3). IR signaling is also involved in 17␤-estradiolmediated neuroprotection in the retina (4). Recent evidence suggests a down-regulation of IR kinase activity in diabetic retinopathy that is associated with the deregulation of downstream signaling molecules (5). Deletion of several downstream effector molecules of the IR signaling pathway, such as IRS-2 (6), Akt2 (7), and Bcl-xl (8), in the retina resulted in a photoreceptor degeneration phenotype. These studies clearly indicate the importance of the IR signaling pathway in the retina.The IR is highly conserved, and the high degree of IR signaling homology between Caenorhabditis elegans, Drosophila, and humans suggests functional conservation in mammalian retina. The IR regulates neuronal survival in C. elegans (9). In Drosophila, the IR serves an important function to guide retinal photoreceptor axons from the retina to the brain during development (10), and the IR influences the size and number of photoreceptors (11). The lack of IR activation leads to neurod...

In the present study, we tested the hypothesis that 17␤-estradiol (␤E2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H 2 O 2 )-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both ␤E2 and 17␣-estradiol (␣E2) significantly protected against H 2 O 2 -induced retinal neuron degeneration; however, progesterone had no effect. ␤E2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [32 ␥ATP] were used as substrate. Phospho-Akt levels were also transiently increased by ␤E2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of ␤E2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of ␤E2, suggesting that ␤E2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that ␤E2 and ␣E2, but not progesterone, identified phosphorylated insulin receptor ␤-subunit (IR␤) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IR␤ activation of PI3K. Systemic administration of ␤E2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of ␤E2 activated retinal IR␤, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.