Functional characterization in vitro of twelve naturally occurring variants of the human pancreatic polypeptide receptor NPY4R

• Obesity has become a global health problem and therefore understanding of the mechanisms regulating hunger and satiety is of utmost importance for the development of new treatment strategies. The Y4 receptor, encoded by the NPY4R gene, and its ligand pancreatic polypeptide (PP) have been reported to mediate a satiety signal. Multiple genetic studies have reported an association between NPY4R copy number and body weight. The gene also displays several SNP variants, many of which lead to amino acid differences, making it interesting to study. We have investigated the functional properties of 12 naturally occurring amino acid sequence variants of the Y4 and interpret the results in relation to sequence conservation and our structural model of the human Y4 receptor protein. Three receptor variants, Cys201ECL2Tyr, Val2716.41Leu and Asn3187.49Asp, were found to completely lose functional response, measured as inositol phosphate turnover, while retaining membrane expression. They display high sequence conservation and have important roles in the receptor structure. For two receptor variants the potency of PP was significantly decreased, Cys34NTSer (EC50 = 2.9 nM, p < .001) and Val1353.46Met (EC50 = 3.0 nM, p < .01), compared to wild-type Y4 (EC50 = 0.68 nM). Cys34 forms a disulphide bond with Cys298, linking the N-terminal part to ECL3. The Val1353.46Met variant has an amino acid replacement located in the TM3 helix, one helix turn above the highly conserved ERH motif. This position has influence on the network of residues involved in receptor activation and subsequent inactivation. Sequence conservation and the structural model are consistent with these results. The remaining seven positions had no significant effect on the receptor's functional response compared to wild-type Y4. These positions display more variation during evolution. Understanding of the interactions between the Y4 receptor and its native PP agonist and the effects of amino acid variation on its functional response will hopefully lead to future therapeutic possibilities.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Y4

SNP

Mutagenesis

Functional pharmacology

Structural modelling

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