Associate editor: J. Wess Fluorescent ligands, antibodies, and proteins for the study of receptors Craig J. Daly * , John C. McGrath Faculty of Biomedical and Life Sciences, Division of Neuroscience and Biomedical Systems, University of Glasgow, Wolfson Building (Office 448), West Medical Building (Lab 440), University Avenue, Glasgow G12 8QQ, UK Abstract Fluorescent molecules bound to receptors can show their location and, if binding is reversible, can provide pharmacological information such as affinity and proximity between interacting molecules. The spatial precision offered by visualisation transcends the diverse localisation and low molecular concentration of receptor molecules. Consequently, the relationships between receptor location and function and life cycles of receptors have become better understood as a result of fluorescent labeling. Each of these aspects contributes new insights to drug action and potential new targets. The relationships between spatial distribution of receptor and function are largely unknown. This is particularly apparent for native receptors expressed in their normal host tissues where communication between heterogeneous cell types influences receptor distribution and function. In cultured cell systems, particularly for G-protein-coupled receptors (GPCR), fluorescence- based methods have enabled the visualisation of the cycle of agonist-stimulated receptor clustering, endocytic internalisation to the perinuclear region, degradation of the receptor-ligand complex, and recycling back to the surface membrane. Using variant forms of green fluorescent protein (GFP), antibodies, or fluorescent ligands, it is possible to detect or visualise the formation of oligomeric receptor complexes. Careful selection of fluorescent molecules based on their spectral properties enables resonance energy transfer and multilabel visualisation with colocalisation studies. Fluorescent agonist and antagonist ligands are now being used in parallel with GFP to study receptor cycling in live cells. This review covers how labeling and visualisation technologies have been applied to the study of major pharmacologically important receptors and illustrates this by giving examples of recent techniques that have relied on GFP, antibodies, or fluorescent ligands alone or in combination for the purpose of studying GPCR. D 2003 Elsevier Inc. All rights reserved. Keywords: Fluorescence; Fluorescence resonance energy transfer; Green fluorescent protein; Antibodies; Receptors; Imaging Abbreviations: Ach, Acetylcholine; AR, Adrenergic Receptor; BODIPY, bordifluoropyrromethene; CCK, cholecystokinin; CHO, Chinese Hamster Overy; CLSM, Confocal Laser Scanning Microscope; FITC, Fluorescein Isothiocyanate; FLIM, Fluorescence Lifetime Imaging; FRAP, Fluorescence Recovery After Photobleaching; FRET, Fluorescence Energy Transfer; GFP, Green Fluorescent Protein; GPCR, G Protein Coupled Receptor; HEK, Human Embrionic Kidney; IHC, Immunohistochemistry; NA, Noradrenaline or Numerical Aperture; NADH, Nicotinamide Adenine Dinucleotide; NPY, Neuropeptide Y; NT, Neurotensin; OT, Oxytocin; PI, Phosphatidylinositol; PMT, Photomultiplier Tube; PR, Progesterone Receptor; PSF, Point Spread Function; QAPB, Quinazoline Piperazine Bodipy; RI, Refractive Index; SMC, Smooth Muscle Cell; SP, Substance P; SSTR, Somatostatin Receptor; TMR, Tetra Methyl Rhodamine; TSA, Tyramide Signal Amplification; UV, Ultra Violet; VIP, Vasoactive Intestinal Polypeptide. Contents 1. Introduction ............................................ 102 2. Fluorescence ........................................... 103 2.1. What is fluorescence? ................................... 103 2.2. The early use of fluorescence ............................... 104 3. Fluorescence related to receptor biology ............................. 104 3.1. Imaging receptors ..................................... 105 3.2. Immunohistochemistry ................................... 106 3.3. Fluorescent ligands .................................... 106 3.3.1. Physics ...................................... 107 0163-7258/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2003.08.001 * Corresponding author. Tel.: +44-141-330-3920; fax: +44-141-330-2923. E-mail address: c.daly@bio.gla.ac.uk (C.J. Daly). www.elsevier.com/locate/pharmthera Pharmacology & Therapeutics 100 (2003) 101 – 118