Many G protein coupled receptors are phosphorylated after their activation. As a consequence they bind ß-arrestin, preventing G protein interaction and targeting the receptors to clathrin coated pits (Ferguson, 2001). The Y₁ receptor, responding to neuropeptide Y (NPY) and peptide YY (PYY), undergoes desensitisation and endocytosis (Gicquiaux et al., 2002) and associates with ß-arrestin 2 (Berglund et al., 2003). Here we compare native or haemaggluttinin (HA)-tagged rat Y₁ receptors with two truncated mutants (T361* and S352*) to begin to establish the C terminal regulatory motifs that underly their desensitisation.

Y1 receptor mutagenesis was performed with the Transformer kit (Clontech), and stable Chinese hamster ovary (CHO) or Colony 1 epithelial clones were selected by neomycin resistance. [¹²⁵I]PYY and GTP³⁵S binding assays, and measurements of epithelial chloride secretion (as short-circuit current, I_SC) were performed as described (Holliday & Cox, 2003). In desensitisation experiments, CHO clones were pretreated at 37°C with 100 nM NPY or vehicle for 10 min and washed extensively, before membrane preparation for GTP³⁵S assays at 21°C (90 min NPY (0.03-1000 nM), followed by 20 min 200 pM GTP³⁵S incubation; in 50 mM NaCl and 1 µM GDP buffer). Non-cumulative concentration response-curves yielded EC₅₀ values (Graphpad Prism v3.01); significant data groups were determined by Student's t-test.

In GTP³⁵S assays, CHO clones expressing HA-Y₁, HA-Y₁(S352*) or HA-Y₁(T361*) receptors ([¹²⁵I]PYY saturation pK_i: 9.32-9.52, B_max 3.9-5.6 pmol mg_-1; n = 4-5) responded to NPY with similar EC₅₀ values (1.4-4.6 nM) and response to 1 µM agonist (198 - 234 % of basal binding (205 - 251 fmol mg_-1); n = 4-5). In NPY pretreated cells the inhibition of basal GTPg³⁵S binding by the Y₁ antagonist BIBO3304 (1µM) was unaltered (e.g. in HA-Y₁ 3.9±3.7 vs 3.8±2.6 % in untreated, n = 5), confirming removal of initial NPY. However 1µM NPY responses were reduced in membranes from pretreated HA-Y₁ (142.1 ± 1.7 vs 197.8 ± 6.9 % in control; P < 0.001; n = 5) and HA-Y1(T361*) cells (142.4 ± 8.9 vs 202.0 ± 11.6 %; P < 0.01; n = 4 - 5), while they were less affected in the HA-Y₁(S352*) clone (190.3 ± 10.5 vs 234.6 ± 16.0 %; n = 4).

PYY inhibited vasoactive intestinal polypeptide stimulated ISC in Y₁, Y₁(S352*) and Y₁(T361*) Colony 1 clones (EC₅₀: 18.2 - 29.5 nM; n = 3 - 5), with respective 100 nM responses being -10.0 ± 1.9, -5.4 ± 0.9 and -5.2 ± 0.8 µA cm-2 (n = 5 -8). In contrast to the transient Y₁ and Y₁(T361*) responses (100 nM PYY at 10 min, -20.7 ± 5.3 % and -24.5 ± 10.4 % of peak ISC inhibition at 2 - 3 min), Y₁(S352*) responses were sustained (-61.0 ± 11.9 %, P < 0.05 compared to Y₁ control). We conclude that S352* truncation does not inhibit Y₁ receptor G protein coupling, but the sequence S352 - T361 (including 4 potential S/T phosphorylation sites) forms a key element controlling its desensitisation.

Berglund, MM et al.(2003) J.Pharmacol.Exp.Ther. 306,147-56.
Ferguson, SSG (2001) Pharmacol.Rev. 53, 1-24.
Gicquiaux, H et al. (2002) J.Biol.Chem. 277, 6645-55.
Holliday, ND & Cox, HM (2003) Br.J.Pharmacol. 139,501-12.

Supported by the BBSRC and the Kimmel Cancer Foundation.