Holliday, Nicholas Dargue; (1998) Functional expression of native and mutant Y1 receptors in stably transfected adenocarcinoma cell lines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Neuropeptide Y (NPY) and peptide YY (PYY) are potent antisecretory agonists in the gastrointestinal tract, attenuating electrogenic chloride and fluid secretion in vitro and in vivo in various species. Human adenocarcinoma cell lines provide model systems in which to investigate the mechanisms by which epithelial Y receptors modulate anion transport, in the absence of other mucosal cell types. Two of these (HT-29 and HCA-7 Colony 1) do not possess PYY receptors and were consequently stably transfected with the cDNA encoding the Y1 subtype, endogenously expressed in human colonic mucosa. Both wild type epithelia and the Y1 clones responded to a similar range of secretory and antisecretory agents when voltage-clamped in Ussing chambers; in addition, PYY transiently reduced both basal and cAMP-stimulated short-circuit current in Y1 epithelial layers. These antisecretory responses were inhibited by the Y1 antagonists BIBP 3226 and GR231118. Surprisingly, PYY and [Leu31, Pro34] (Pro34) PYY were 10-20 fold more potent than NPY or Pro34NPY in both HT-29 and Colony 1 clones, despite each agonist displaying equivalent binding affinities. It is suggested that metabolism by membrane surface peptidases may underlie this selectivity, and that this may influence responses at native mucosal Y1 receptors. The Colony 1 Y1 clone was also compared with those transfected with Y1 receptor cDNAs containing single amino acid mutations. The Y1(S255E) receptor was apparently down-regulated, with associated consequences for PYY antisecretory responses; phosphorylation of Ser255 in the third intracellular loop (imitated by Glu) may therefore be important in desensitisation. Substitution of Cys337, potentially palmitoylated, did not alter receptor expression or the functional potency of PYY. However Y1(C337S) mediated responses remained sustained at high agonist concentration, indicating that Y1 receptor depalmitoylation may inhibit its inactivation. Epithelial expression of receptor mutants may therefore be valuable in assessing their functional properties, particularly regarding desensitisation.

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