RHODOPSIN MUTANTS

受体磷酸化和棕榈酰化是两种修饰 在G蛋白家族中的许多受体的C末端 偶联受体（GPCR）。 这两种修饰的流行程度 表明了它在受体功能中的重要作用。磷酸化 受体通常对通过配体结合的激活不太敏感。 然而，脱敏中磷酸化机制的细节 不知道。 棕榈酰化在不同受体中的作用是 不太清楚。信号转导在一些受体中受到影响， 受体内化、螯合和磷酸化可能是 在其他受体中单独或以各种组合受到影响。 我们建议研究磷酸化和棕榈酰化对 视紫红质分子中的受体功能。为此，我们 制造了视紫红质基因敲除小鼠来研究磷酸化， 棕榈酰化缺陷型视紫红质突变体。 具体而言，我们将（i） 完成视紫红质半合子和纯合子的鉴定 敲除小鼠; ii）测试不同的视紫红质 磷酸化位点对受体具有不同的生理作用 敏感性和iii）测试视紫红质棕榈酰化 在调节受体对光刺激的反应中起作用。 突变的视紫红质转基因将用于产生转基因小鼠， 与视紫红质敲除小鼠杂交。 视网膜形态将是 通过光学显微镜检查，生理功能将被 通过光诱发光反应的单细胞记录来评估。 将通过以下方法评估突变对蛋白定位的影响： 免疫组化 有趣的是，视紫红质基因C端的突变 与遗传性人类视网膜变性疾病有关， 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以深入了解 导致受体功能障碍和疾病的缺陷。

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.