Structure of the Human Cannabinoid Receptor

描述(由申请人提供)： 这项建议的主要目的是了解螺旋束的结构，螺旋束组成人大麻素受体(CB1)的完整膜部分和配体结合部位。了解束内的螺旋-螺旋相互作用将有助于阐明配体结合口袋中涉及的残基，这一信息对治疗学的进一步发展至关重要。该提案的目的是识别驱动CB1从片段中组装的跨膜结构域，并识别参与这些相互作用的螺旋区域。在HEK 293细胞中表达的受体将结合特定的蛋白酶识别的位点，并被蛋白水解性切割以产生大片段。天然形式的保留将通过配体结合和G蛋白激活分析来确定，并将用于鉴定片段之间稳定的螺旋间联系。通过半胱氨酸二硫化物的交联，相互作用的表面将被更精确地映射。这些研究将提高我们对相互作用的跨膜片段如何排列在螺旋束中以协调受体组装、配体结合和受体激活的理解。然后，这些信息可以用于开发选择性疗法和药物，以对抗大麻的不良影响。

DESCRIPTION (provided by applicant): The main objective of this proposal is to learn about the structure of the helical bundle, which makes up the integral membrane portion and the ligand-binding site of the human cannabinoid receptor (CB1). Understanding the helix-helix interactions within the bundle will help to elucidate the residues involved in the ligand-binding pocket, information that is crucial for further development of therapeutics. The aims of the proposal are to identify transmembrane domains that drive the assembly of CB1 from fragments, and to identify helical regions involved in these interactions. Receptors will be generated that incorporate sites for recognition by specific proteases, expressed in HEK 293 cells and proteolytically cleaved to produce large fragments. Retention of the native form will be determined by ligand binding and G protein activation assays and will serve to identify stabilizing interhelical associations between the fragments. The interacting surfaces will be more precisely mapped via cysteine disulfide crosslinking. These studies will improve our understanding of how interacting transmembrane segments are arranged in the helical bundle in order to orchestrate receptor assembly, ligand binding and receptor activation. This information can then be applied to the development of selective therapeutics and drugs to antagonize the ill effects of marijuana.