Intracellular Signaling In Endocrine Cells

该项目涉及内分泌和神经内分泌细胞中的细胞信号级联，以及质膜电事件和受体介导的细胞内信号和分泌之间的相互作用。目前的重点是垂体细胞中表达的嘌呤能受体的特征及其脱敏的潜在机制。这些细胞分泌三磷酸腺苷，作为两个嘌呤能受体家族的自分泌和/或旁分泌细胞外信使，称为P2YRs和P2XRs。前者是G蛋白偶联受体，而P2XRs是阳离子通透性通道，如果在可兴奋细胞中表达，它可以传导钙并促进电压敏感性钙进入。这些通道在激活、脱敏和恢复过程中的配基偏好和通道动力学方面各不相同。然而，不同的受体亚域对亚型特异性行为的贡献还没有完全确定。我们最近发现野生型P2X2aR及其剪接形式缺乏细胞内Val370-Gln438 C末端序列(P2X2bR)，对ATP刺激的反应具有类似的EC50和峰值电流/钙反应，但以受体特异性的方式减敏：P2X2aR减敏缓慢，P2X2bR迅速减敏。 我们研究了不同激动剂和取代胞外结构域对P2X2aR和P2X2bR钙信号转导模式的影响。两种受体对激动剂表现出相似的EC50和IC50，顺序为：2-MES-ATP&lt；ATP&lt；ATP-g-S&lt；bzATP&lt；ab-亚甲基ATP；激动剂的IC50较其EC50右移。此外，ATP诱导的受体亚型特异性脱敏模式可以被高效激动剂模仿，但不能被低效激动剂模仿，这表明了一种配体特异性脱敏模式。为了验证这一假设，我们构建了包含P2X3R的Val60-Phe301外区序列和P2X7R的Val61-Phe313外区序列的嵌合P2X2aR和P2X2bR，而不是天然的Ile66-Tyr310序列。在以P2X2a为基础的骨架中，具有P2X3亚单位胞外结构域的同源受体模拟了P2X3R的两个内在功能，即对α-亚甲基ATP的敏感性和依赖外源ATPase的内源性脱敏恢复；这两个功能分别定位于P2X3R胞外环的N端和C端。此外，突变的P2X2a+X3R和P2X2b+X3R对亚微摩尔浓度范围内的ATP、BzATP和亚甲基ATP显示出相似的EC50，并且以受体特异性和配体非特异性的方式脱敏。另一方面，嵌合的P2X2+X7R对ATP的敏感性降低，并以受体非特异性的方式脱敏。这些结果表明，激动剂对P2X2Rs配体结合域的有效性反映了其C-末端结构控制的脱敏强度。最后，我们分析了异构体中P2XRs的激动剂选择性和脱敏率。首先，我们演示了天然和突变的P2X2a和P2X2b亚基的物理和功能异构化。在异构体受体中，P2X3的胞外区是配体选择性和脱敏回收率的结构决定因素，而P2X2R的C末端是脱敏速率的重要因素。此外，光反应激动剂[Gamma-32P]8-叠氮基ATP在同聚体受体中能有效地与P2X3亚基发生交联，而在异构体受体中则不能。这些结果表明，由不同的P2XR亚基形成的异构体受体在参与的胞外亚单位和C末端亚域的整合作用下发挥新的功能。

This project addresses the cellular signaling cascade in endocrine and neuroendocrine cells, and the interactions between plasma membrane electrical events and receptor-mediated intracellular signaling and secretion. Current emphasis is on the characterization of purinergic receptors expressed in pituitary cells and the underlying mechanism of their desensitization. These cells secrete ATP, which acts as an autocrine and/or paracrine extracellular messenger on two families of purinergic receptors termed P2YRs and P2XRs. The former are G protein-coupled receptors and P2XRs are cation-permeable channels that conduct calcium and facilitate voltage-sensitive calcium entry if expressed in excitable cells. These channels differ among themselves with respect to their ligand preferences and channel kinetics during activation, desensitization and recovery. However, the contributions of distinct receptor subdomains to the subtype-specific behavior have been incompletely characterized. We recently showed that the wild-type P2X2aR and its splice form lacking the intracellular Val370-Gln438 C-terminal sequence (P2X2bR) respond to ATP stimulation with comparable EC50s and peak current/calcium responses, but desensitize in a receptor-specific manner: P2X2aR desensitize slowly and P2X2bR desensitize rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X2aR and P2X2bR. Both receptors showed similar EC50s and IC50s for agonists, in the order: 2-MeS-ATP < ATP < ATP-g-S < BzATP << ab-methylene ATP, and the IC50s for agonists were shifted to the right compared to their EC50s. Furthermore, the ATP-induced receptor-subtype specific pattern of desensitization was mimicked by high- but not by low-efficacy agonists, suggesting a ligand-specific desensitization pattern. To test this hypothesis, we generated chimeric P2X2aR and P2X2bR containing the Val60-Phe301 ectodomain sequence of P2X3R and Val61-Phe313 ectodomain sequence of P2X7R instead the native Ile66-Tyr310 sequence. The homomeric receptors having the extracellular domain of P2X3 subunit in the P2X2a-based backbone mimicked two intrinsic functions of P2X3R, sensitivity to ab-methylene ATP and ecto-ATPase-dependent recovery from endogenous desensitization; these two functions were localized to the N- and C-terminal halves of the P2X3R extracellular loop, respectively. Furthermore, the mutated P2X2a+X3R and P2X2b+X3R exhibited comparable EC50s for ATP, BzATP, and ab-methylene ATP in the submicromolar concentration range, and desensitized in a receptor-specific and ligand-nonspecific manner. On the other hand, the chimeric P2X2+X7R exhibited decreased sensitivity for ATP and desensitized in a receptor-nonspecific manner. These results suggest that efficacy of agonists for the ligand-binding domain of P2X2Rs reflects the strength of desensitization controlled by their C-terminal structures. Finally, we have analyzed the agonist selectivity and desensitization rates of P2XRs in heteromeric configuration. First, we demonstrated the physical and functional heteromerization of native and mutant P2X2a and P2X2b subunits. In heteromeric receptors, the ectodomain of P2X3 was a structural determinant for ligand selectivity and recovery from desensitization, and the C-terminus of P2X2R was an important factor for desensitization rate. Furthermore, [gamma-32P]8-azido ATP, a photoreactive agonist, was effectively cross-linked to P2X3 subunit in homomeric receptors but not in heteromeric P2X2+P2X3Rs. These results indicate that heteromeric receptors formed by distinct P2XR subunits develop new functions resulting from integrative effects of the participating extracellular and C-terminal subdomains.