Intracellular Signaling In Endocrine Cells

Child Health And Human Development

Investigators

Linked publications & trials

Abstract

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.