Analysis of the importance of two PH domains of centaurin-αl for P1P3 binding

Abstract

Constructs of the 5-HT tag for either haemagglutinin (HA) or protein-C (PrC) on the Nterminus of the receptor, or Cyan-Fluorescent Protein (CFP) on the C-terminus. C O S 7 cells were transiently transfected using Fugene 6 and expression of the tagged receptors was assessed by homologous displacement of ['Hlketanserin binding. The 5-HT,,-CFP receptor was expressed most highly then HA-and lastly the PrC-5-HTZA receptor (630,240, and 170 fmol/mg protein respectively; wild type receptor was 440 fmol/mg protein). When the tagged receptors were immunoprecipitated using specific anti-tag antibodies, and visualised on Western blot, the HA-5-HT2, receptor was barely detectable. The PrC-5-HT2, receptor ran as 2 broad bands of 55-65 kDa and 75-90 kDa, whereas the 5-HT2,-CFP receptor ran as a major band of 110-140 with lesser bands at 85,70,62 and 55 kDa. Production of 5-HT-induced [3H]inositol phosphates was measured to assess the function of the tagged receptors. We conclude that the PrC-and the -CFP tagged 5-HTz, receptor constructs will be useful tools to examine receptor-protein interactions.receptor were made with an epitope 2 A