FLUORESCENT-LABELED ANALOGS OF NEUROPEPTIDE-Y FOR THE CHARACTERIZATION OF CELLS EXPRESSING NPY RECEPTOR SUBTYPES

Porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreati c polypeptide family, and subtype selective analogues have been synthe sized by solid phase peptide synthesis. The peptides were labelled wit h Cy3(TM), a commercially available fluorescent marker based on a cyan ine dye, by solid phase strategy. During the cleavage a partial fragme ntation of the fluorescent marker occurred. This has been investigated by means of HPLC and electrospray mass spectrometry. The labelled ana logues of NPY showed high affinity to the NPY receptor subtypes Y-1 an d Y-2. Thus, Cy3-NPY, Y-1-selective Cy3-[Pro(34)] NPY and Y-2 selectiv e Cy3-[Ahx(5-24)] NPY were used to label SK-N-MC- and SMS-KAN-cells, w hich are stably expressing the Y-1-(SK-N-MC) and the Y-2-receptor subt ype (SMS-KAN). The binding of the labelled analogues to the receptors was reversible and specific. The photoactivatable analogue, [(Tmd)Phe( 27)] NPY, which showed high affinity to both receptor subtypes was lab elled with Cy3 in solution. Whereas the fluorescent labelling of the c ells with analogues without photoactivatable amino acid was reversible , successful photocrosslinking could be investigated by the irreversib le staining of the cells using Cy3-[(Tmd)Phe(27)] NPY. These subtype s elective analogues are exciting tools to trace receptors in tissues an d to identify the pharmacologically characterized subtypes without rad ioactivity.