RHODOPSIN MUTANTS

受体磷酸化和棕榈酰化是两种修饰 在G蛋白家族中一些受体的C末端 偶联受体(GPCRs)。这两个修改的流行程度 提示在受体功能中起着重要作用。磷酸化 受体通常对配体结合的激活不那么敏感。 然而，脱敏过程中的磷酸化机制的细节 都是未知的。棕榈酰化在不同受体中的作用是 不太清楚。信号转导在某些受体中受到影响，而 受体内化、隔离和磷酸化可能是 在其他受体中单独或以不同的组合影响。 我们建议研究磷酸化和棕榈酰化在 视紫红质分子中的受体功能。为此，我们有 产生了视紫红质基因敲除小鼠，在其中研究磷酸化-和 棕榈酰化缺陷视紫红质突变体。具体来说，我们将i) 视紫红质半合子和纯合子的完整特征 基因敲除小鼠；ii)检验不同视紫红质的假设 磷酸化位点对受体有不同的生理作用 敏感性和iii)检验视紫红质棕榈酰化的假设 在调节感受器对光刺激的反应中起作用。 突变的视紫红质转基因将用于生产转基因小鼠和 与视紫红质基因敲除小鼠杂交。视网膜形态将会是 通过光学显微镜检查，生理功能将被 通过光引起的光反应的单细胞记录进行评估。 突变对蛋白质定位的影响将通过以下方式进行评估 免疫组织化学。 有趣的是，视紫红质基因C末端的突变 与遗传性人类视网膜退行性疾病有关， 视网膜色素变性。阐明C-末端结构域功能 通过磷酸化和棕榈酰化修饰可能会提供对 缺陷会导致受体功能障碍和疾病。

Receptor phosphorylation and palmitoylation are two modifications seen in C-terminus of a number of receptors in the family of G-protein coupled receptors (GPCRs). The prevalence of these two modifications suggests an important role in receptor function. Phosphorylated receptors are typically less sensitive to activation by ligand binding. However, the details of the phosphorylation mechanism in desensitization are not known. The role of palmitoylation in different receptors is less clear. Signal transduction is affected in some receptors, while receptor internalization, sequestration and phosphorylation may be affected singly or in various combination in other receptors. We propose to examine the role of phosphorylation and palmitoylation on receptor function in the rhodopsin molecule. Towards this end, we have produced rhodopsin knockout mice in which to study phosphorylation- and palmitoylation-defective rhodopsin mutants. Specifically, we will i) complete the characterization of rhodopsin hemizygous and homozygous knockout mice; ii) test the hypothesis that different rhodopsin phosphorylation sites have different physiological effects on receptor sensitivity and iii) test the hypothesis that rhodopsin palmitoylation has a role in regulating the receptor response to light stimulation. Mutant rhodopsin transgenes will be used to produce transgenic mice and cross-bred with rhodopsin knockout mice. Retinal morphology will be examined by light microscopy, and physiological function will be assessed by single cell recordings of the light evoked photoresponse. Effects on protein localization of the mutation will be assessed by immunohistochemistry. Interestingly, mutations in the C-terminus of the rhodopsin gene have been associated with the inherited human retinal degenerative disease, retinitis pigmentosa. Elucidating C-terminal domain functions that are modified by phosphorylation and palmitoylation may provide insight into defects leading to receptor dysfunctions and disease.