Human urotensin-II receptor desensitisation

Human Urotensin-II (U-II) is a cyclic undecapeptide that binds to the U-II receptor UT. The desensitisation mechanisms of the UT receptor (G_{q/11} coupled GPCR) are not well defined and hampered by (1) lack of native (in-vitro) models; (2) paucity of ligands, especially non-peptides and (3) irreversible binding of U-II. There are some limited studies using rat aorta, where a U-II induced primary contractile response was reduced upon a secondary re-challenge after 5-hours. Studies were undertaken to characterise cell lines expressing native (SJCRH30) and recombinant human hUT (HEK293 and CHO) for their suitability in binding and functional assays (PI and Ca^2+). SAR studies were carried out to characterise novel analogues modified at Tyr^9 of the U-II(4-11) template. This led to the identification of [3,5-diiodoTyr^9]U-II(4-11) a partial agonist in aorta and Ca^2+ assays at rat UT. Full agonism was demonstrated at hUT in PI and Ca^2+ assays. Efforts were made to delineate functional and genomic desensitisation of hUT. There was no functional desensitisation in SJCRH30. In HEK293hUT functional heterologous desensitisation of hUT was observed, this was not so in CHOhUT; instead P_2YR was functionally attenuated. In SJCRH30 6-hr U-II treatments led to UT mRNA reduction. Genomic desensitisation was also studied in Peripheral blood mononuclear cells (PBMCs). U-II treatments alone did not affect UT mRNA. Lipolysaccharide treatment of PBMCs led to UT mRNA upregulation which was desensitised with U-II treatments. In recombinant systems UT mRNA was upregulated at 6-hr U-II treatments. In conclusion modification of the U-II(4-11) template at Tyr^9 is useful for reducing efficacy. There is a difference in desensitisation profiles of native and recombinant hUT, where native receptors are not prone to functional desensitisation while receptor mRNA is reduced. In recombinant systems, hUT undergoes desensitisation (HEK293hUT only) while receptor mRNA is increased in both systems.