ß2-Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function.

Bitter taste receptors (TAS2Rs) are G-protein-coupled receptors now recognized to be expressed on extraoral cells, including airway smooth muscle (ASM) where they evoke relaxation. TAS2Rs are difficult to express in heterologous systems, with most receptors being trapped intracellularly. We find, however, that co-expression of ß2-adrenergic receptors (ß2AR) in HEK-293T routes TAS2R14 to the cell surface by forming receptor heterodimers. Cell surface TAS2R14 expression was increased by ∼5-fold when ß2AR was co-expressed. Heterodimer formation was shown by co-immunoprecipitation with tagged receptors, biomolecular fluorescence complementation, and merged confocal images. The dynamic nature of this interaction was shown by: a gene-dose relationship between transfected ß2AR and TAS2R14 expression, enhanced (up to 3-fold) TAS2R14 agonist stimulation of [Ca(2+)]i with ß2AR co-transfection, ∼53% decrease in [Ca(2+)]i signaling with shRNA knockdown of ß2AR in H292 cells, and ∼60% loss of [Ca(2+)]i responsiveness in ßAR knock-out mouse ASM. Once expressed on the surface, we detected unidirectional, conformation-dependent, interaction within the heterodimer, with ß2AR activation rapidly uncoupling TAS2R14 function (∼65% desensitization). Cross-talk was independent of ß2AR internalization and cAMP/PKA, and not accompanied by TAS2R14 internalization. With prolonged ß-agonist exposure, TAS2R14 internalized, consistent with slow recycling of naked TAS2R14 in the absence of the heterodimeric milieu. In studies of ASM mechanics, rapid cross-talk was confirmed at the physiologic level, where relaxation from TAS2R14 agonist was decreased by ∼50% with ß-agonist co-treatment. Thus the ß2AR acts as a double-edged sword: increasing TAS2R14 cell surface expression, but when activated by ß-agonist, partially offsetting the expression phenotype by direct receptor:receptor desensitization of TAS2R14 function.

Sigma-1 receptor: the novel intracellular target of neuropsychotherapeutic drugs.

Sigma-1 receptor ligands have been long expected to serve as drugs for treatment of human diseases such as neurodegenerative disorders, depression, idiopathic pain, drug abuse, and cancer. Recent research exploring the molecular function of the sigma-1 receptor started unveiling underlying mechanisms of the therapeutic activity of those ligands. Via the molecular chaperone activity, the sigma-1 receptor regulates protein folding/degradation, ER/oxidative stress, and cell survival. The chaperone activity is activated or inhibited by synthetic sigma-1 receptor ligands in an agonist-antagonist manner. Sigma-1 receptors are localized at the endoplasmic reticulum (ER) membranes that are physically associated with the mitochondria (MAM: mitochondria-associated ER membrane). In specific types of neurons (e.g., those at the spinal cord), sigma-1 receptors are also clustered at ER membranes that juxtapose postsynaptic plasma membranes. Recent studies indicate that sigma-1 receptors, partly in sake of its unique subcellular localization, regulate the mitochondria function that involves bioenergetics and free radical generation. The sigma-1 receptor may thus provide an intracellular drug target that enables controlling ER stress and free radical generation under pathological conditions.

Activation of RidA chaperone function by N-chlorination.

Escherichia coli RidA is a member of a structurally conserved, yet functionally highly diverse protein family involved in translation inhibition (human), Hsp90-like chaperone activity (fruit fly) and enamine/imine deamination (Salmonella enterica). Here, we show that E. coli RidA modified with HOCl acts as a highly effective chaperone. Although activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system and glutathione, it is independent of cysteine modification. Instead, treatment with HOCl or chloramines decreases the amino group content of RidA by reversibly N-chlorinating positively charged residues. N-chlorination increases hydrophobicity of RidA and promotes binding to a wide spectrum of unfolded cytosolic proteins. Deletion of ridA results in an HOCl-sensitive phenotype. HOCl-mediated N-chlorination thus is a cysteine-independent post-translational modification that reversibly turns RidA into an effective chaperone holdase, which plays a crucial role in the protection of cytosolic proteins during oxidative stress.

Glutathione and vitamin B12 cooperate in stabilization of a B12 trafficking chaperone protein.

The protein bCblC (bCblCpro) is a bovine homolog of a human B12 trafficking chaperone that is responsible for the processing of vitamin B12 and its escorted delivery in intracellular B12 metabolism. In this study, we found that bCblCpro is highly thermolabile with a T(m) = 42.0 ± 0.2 °C as shown for the human homolog, suggesting thermal regulation of these proteins. Binding of the reduced form of glutathione (GSH) that is a predominant cellular thiol increased the T(m) of bCblCpro from 42 °C to ~45 °C (ΔT(m max) = 3.1 ± 0.2 °C and AC50 = 2.1 ± 0.5 mM). Binding of vitamin B12 and its derivatives also stabilized bCblCpro increasing the T(m) to a different extent and vitamin B12 (cyanocobalamin, CNCbl) was the least efficient (ΔT(m max) = 4.3 ± 0.3 °C and AC50 = 291 ± 36 µM). However, the stabilizing effect of CNCbl was significantly greater for GSH-bound bCblCpro (ΔT(m max) = 12.8 ± 0.6 °C and AC50 = 9.3 ± 1.6 µM) than for GSH-free bCblCpro. In addition, the stabilizing effect of GSH was also greater for CNCbl-bound bCblCpro (ΔT(m max) = 9.3 ± 0.3 °C and AC50 = 57.0 ± 6.8 µM). Limited proteolysis revealed that thermal stabilization of bCblCpro is derived from conformational changes of the protein induced by binding of the ligands. The results in this study indicate that GSH cooperates with vitamin B12 in thermal stabilization of bCblCpro and is a positive regulator of the protein.