siRNA, demonstrate that only these isoforms are modulating 2C AR temperature dependent trafficking. The two HSP90 cytosolic isoforms BIBR 1532 321674-73-1 are designed and and are closely related, with the most important sequence difference in the N terminus.. Although both isoforms are present under basal conditions, HSP90 usually shows a larger increase after heat shock and therefore is credited BIBR 1532 321674-73-1 to be the inducible isoform, whereas HSP90 which has lesser variations is considered the constitutive isoform. However, each isoform may substitute the other in the cellular functions. Also, the experimental tools to differentiate between the HSP90 isoforms are limited, as the two cytosolic isoforms have similar sensitivity to HSP90 inhibitors, share the same co chaperones, form heterodimers and the antibodies cross react.
Based on these reasons, no attempt was made in the present study to BMS-536924 BMS-536924 differentiate which isoform is crucial for the temperature sensitive 2C AR trafficking. The enhanced 2C AR plasma membrane expression at low temperature and/or after HSP90 inhibition is reflected by increased functional responses after receptor stimulation in these conditions. The classical physiological view attributes all the GPCR function to the receptors present at the cell surface, freely accessible to the extracellular ligands.
However, this paradigm was challenged in the last decade, activation of cellular signaling by receptors with intracellular localization being demonstrated in several situations. However, the large pool of 2C AR localized in the endoplasmic reticulum at physiological temperature appears unable to contribute to cellular responses.
In fact, the effects on cAMP and vascular tone observed at 37 are exclusively due to activation of the receptor fraction with plasma membrane localization, as they are eliminated by addition of the extracellular 2 AR antagonist, rauwolscine. The inability of intracellular 2C AR to trigger cellular signaling may be related to the absence of molecules required to trigger signaling at this level. However, recent data indicate that GPCR are associated in signaling complexes with the corresponding molecules early in the biosynthetic pathway.
More probably, appropriate receptor activators are unable to reach the intracellular 2C AR. Still, our results cannot exclude the possibility that intracellular 2C AR activates other unknown yet signaling mechanisms.
In contrast, when the receptor expression at the cell surface is increased by low temperature and/or HSP90 inhibition, the inhibition of cAMP levels and contractile effects in response to the 2 agonist are markedly enhanced. The similarity of the effects of low temperature and HSP90 inhibition on 2C AR functional responses in HEK293T cells and rat tail artery demonstrate that the temperature sensitive receptor trafficking is not limited to heterologous transfection systems. The effects of low temperature were absent only in PC12, a neuro endocrine cell line, in agreement with previous findings. Different expression of HSP90 isoforms in neurons and in smooth muscle cells have been reported and this fact may explain the cell specific receptor trafficking. The current study reveals a novel aspect of HSP90 inhibitors, specifically modulation of vascular tone. Previously, impairment of th