Characterization of receptors using cyanine 3-labeled neuropeptides.
We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.
D000818:Animals; D002232:Carbocyanines; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003199:Computer Systems; D004705:Endocytosis / Q000502:physiology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D019886:Gastrin-Releasing Peptide; D006168:Guinea Pigs; D015288:Neurokinin A / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D051381:Rats; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism*; D014162:Transfection
D002232:Carbocyanines; D005456:Fluorescent Dyes; D010455:Peptides; D018013:Receptors, Neuropeptide; C089187:cyanine dye 3; D013373:Substance P; D019886:Gastrin-Releasing Peptide; D015288:Neurokinin A