The endothelins (Ets) are the most potent vasoconstrictor peptides known. They are present in mammals and are produced and function actively in almost all tissues. Elevated levels of ET-1 have been reported in numerous diseases. Studies have revealed the existence of multiple receptor subtypes; two of them, designated ETA and ETB receptors, have been extensively investigated and cloned. Pharmacological and physiological data support the existence of additional receptor subtypes.
The carbohydrate moiety of glycoproteins may contribute to their stabilization, secretion, intracellular trafficking, cell-surface expression, folding and functional properties. Elucidation of the molecular mechanisms of endothelin action requires knowledge of the structural components of the receptor and, in particular, their specific roles in endothelin binding. In the case of ET receptors, little is known about the structure or role of their carbohydrate components. It is also not known whether all potential glycosylation sites of the receptors are indeed glycosylated, or whether glycosylation is involved in high-affinity binding and/or G-protein coupling.
An interesting phenomenon observed for both ETA and ETBreceptors is their division to nanomolar-affinity state (high affinity sites) and to picomolar-affinity state (super-high affinity sites). It is not yet known whether these two affinity states represent receptor subtypes ( in which case there would be two subtypes of ETA and two subtypes of ETB receptors) or the same receptor fluctuating between two affinity states.
In this study we investigated whether ligand binding to ET receptors is influenced by the carbohydrate moiety or whether deglycosylation affects merely a subpopulation of ET receptors. It was found that pretreatment of cerebellar or atrial membranes with endoglycosidase H (endo H) caused a marked decrease in the number of maximal binding sites that bind ligand with nanomolar-affinity, whereas ligand affinity remained the same. The picomolar-affinity binding sites were not affected by endo H. The use of specific antagonists indicated that the receptor subtype most likely to be influenced by glycosylation is ETA. Transfection of COS cells with mutated ETA receptor at Asn-29 or Asn 62 revealed that the Asn-62 is important for binding parameters. We suggest that in both cerebellar and atrial membranes, the carbohydrate chains of the ETA receptor contribute to the binding of ligand to the nanomolar-affinity binding sites, but not to the picomolar-affinity binding sites.
