Moreover, other experimental approaches, such as mass spectrometry, may more ac

Internalised receptors may, in turn, then be recycled to the plasma membrane supplier ABT-737 where they are available for another round of signalling or may be targeted for degradation. In the case of the hIP, considerable data has been generated regarding the mechanisms responsible for its phosphorylation, internalisation and recycling. Recent studies have established that Rab5a GTPase plays a critical role in the agonist induced internalisation of the hIP and that Rab4a and Rab11a are involved in its fast and slow recy cling, respectively, to the plasma membrane. However, despite these investigations, little is known about the mechanism regulating the turnover or degra dation of the hIP under either basal or agonist induced conditions. The main systems employed by cells to degrade proteins are the lysosomes and the 26S proteasomes.<br><br> Lyso somes are vesicular organelles containing a variety of pro teases that function in an acidic environment to disrupt peptide bonds. The 26S proteasomes, consisting of a 20S core proteolytic particle and a 19S regulatory particle, generally degrade proteins that have been post transla tionally modified by attachment of the polypeptide buy AEB071 ubiq uitin to the  amino group of Lys residue of the substrate in an ATP dependent manner. It is now apparent that a number of GPCRs can be ubiquitinated. Agonist induced ubiquitination of GPCRs does not appear to target them for degradation by the proteas omes, but is instead required for endocytic sorting of the internalised receptors and, ultimately, may target them for lysosomal degradation.<br><br> On the other hand, ago nist independent ubiquitination of a small number of GPCRs has been identified and is mainly associated purchase AG-014699 with targeting of newly synthesised, misfolded GPCRs in the endoplasmic reticulum for degradation by the cytosolic proteasomes, through a process referred to as ER associated degradation, or ERAD. In this study, it was sought to investigate turnover of the hIP under basal conditions and in response to stimulation with its selective agonist cicaprost. Owing to the relatively low level of endogenous hIP, even within prostacyclin responsive cells of the vasculature, a previously character ised clonal HEK. hIP cell line which stably over expresses hemagglutinin epitope tagged forms of the hIP was used to facilitate these studies.<br><br> Our data establish that the hIP is subject to low levels of degradation under basal conditions but that upon agonist stimulation, its degrada tion is substantially enhanced. Inhibition of the lyso somal pathway prevented basal and agonist induced degradation of the mature hIP. Conversely, while inhibition of the proteasomal degradation pathway had no effect on the level of the mature hIP, it led to a time dependent accumulation of four newly synthesised immature species of the hIP. Data presented herein also suggest that both the mature and immature species of the hIP may be polyubiquitinated and that this modification may be required for lysosomal sorting of mature, internal ised receptors and for degradation of misfolded immature receptors by the 26S proteasomes, respectively.