INTRACELLULAR SIGNALING IN ENDOCRINE CELLS

内分泌细胞中的细胞内信号传导

• 批准号: 6432502
• 负责人: STANKO S. STOJILKOVIC
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432502
• 关键词: adenosine triphosphate; biological signal transduction; calcium channel; calcium flux; gonadotropin releasing factor; hormone receptor; inositol phosphates; luteinizing hormone; nucleotidases; purinergic receptor; receptor coupling; voltage gated channel

This project addresses the cellular signaling cascade in endocrine and neuroendocrine cells and the interactions between plasma membrane electrical events and receptor-mediated calcium signaling. Current emphasis is on the characterization of ATP-gated receptor-channels (P2XR) in these cells. When expressed in excitable GT1 cells, P2XR subtypes differed in the peak amplitudes of their calcium responses independently of the level of receptor expression, with the order: P2X1R < P2X3R < P2X4R < P2X2bR < P2X2aR < P2X7R. During prolonged agonist stimulation, calcium signals desensitized with different rates: P2X3R > P2X1R > P2X2bR > P2X4R >> P2X2aR >> P2X7R. A slow desensitizing pattern of P2X2aR was mimicked partially by P2X3R and fully by P2X4R mutants in which six amino acid (6-aa) sequences in their C-termini were substituted with the corresponding arginine 371 to proline 376 sequence of P2X2aR. Changing the total net charge in the 6-aa sequence of P2X4R to a more positive direction also slowed receptor desensitization. On the other hand, substitution of the arginine 371 - proline 376 sequence of P2X2aR with the corresponding sequences of P2X1R, P2X3R, and P2X4R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X2aR and mutant P2X3R having the C-terminal 6-aa of P2X2aR at its analogous position resulted in a functional channel, with significantly delayed desensitization. The P2X1R, P2X3R, and P2X4R-derived calcium signals were predominantly dependent on activation of voltage-sensitive calcium influx, whereas voltage-sensitive and -insensitive entry pathways equally contributed to cytosolic calcium responses in P2X2aR-, P2X2bR- and P2X7R-expressing cells. The transcripts and functional P2X7R, P2X3R, and P2X4R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Co-expression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X2aR and P2X2bR, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. These results indicate that calcium signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific. This provides an effective mechanism for generating variable calcium patterns in response to a common agonist.

该项目解决了内分泌和神经内分泌细胞中的细胞信号传导级联，以及质膜电动事件与受体介导的钙信号传导之间的相互作用。 当前的重点是这些细胞中ATP门控受体通道（P2XR）的表征。 当在可激发的GT1细胞中表达时，P2XR亚型在其钙反应的峰值幅度上有所不同，独立于受体表达水平，其顺序为：p2x1r <p2x3r <p2x4r <p2x4r <p2x2x2bbr <p2x2br <p2x2br <p2x2ar <p2x7r。 在长时间的激动剂刺激期间，钙信号以不同的速率脱敏：p2x3r> p2x1r> p2x2br> p2x4r >> p2x2ar >> p2x7r。 P2X3R部分模仿P2X2AR的缓慢脱敏模式，并通过P2X4R突变体完全模仿其C-Termini中的六个氨基酸（6-AA）序列，用相应的精氨酸371代替P2X2AR的脯氨酸376序列。将P2X4R的6-AA序列中的总净电荷更改为更积极的方向也减慢了受体脱敏。 另一方面，用P2X1R，P2X3R和P2X4R的相应序列取代精氨酸371-脯氨酸376序列P2X2AR序列增加了受体脱敏的速率。此外，野生型P2X2AR和突变体P2X3R的异源聚合具有P2X2AR的C末端6-AA，其类似位置会导致功能通道，并具有明显延迟的脱敏。 P2X1R，P2X3R和P2X4R衍生的钙信号主要取决于电压敏感的钙涌入的激活，而电压敏感和不敏感的入口途径同样有助于P2X2AR-，P2X2AR-，P2X2BBR-和P2XX7R- p2x2br-和P2X7R- p2X2AR-，P2X2AR-，P2X2AR-钙反应。 转录本和功能性P2X7R，P2X3R和P2X4R在乳营养和GH3细胞中鉴定出来，但在淋巴瘤和促性腺营养嗜酸群中却没有鉴定。 在74％的乳酸营养物中鉴定出功能性P2X7R，而这些细胞中有50％表达P2X3R和33％表达P2X4R。 经常观察到这些受体亚型的共表达。 纯化的人营养噬菌体表达了P2X2AR和P2X2BR的转录本，并且在体营养和促性腺营养中鉴定出功能受体，但在乳营养营养不良。 这些结果表明，在可激发细胞中表达的同源P2XR的钙信号传导是亚型特异性的。 这提供了一种有效的机制来生成可变的钙模式，以响应共同的激动剂。