Synthesis and properties of three fluorescent derivatives of gastrin

Abstract

As a first step in the study of hormone interaction with gastrin receptor-expressing cells, three fluorescent derivatives of heptagastrin were synthesized, characterized and tested for specificity and affinity towards gastrin/ CCKB receptor by means of confocal laser scanning microscopy (CLSM). Cyanine dye Cy3.29 and borfluoropyrromethene (BODIPY) derivatives of the hormone were found to be absorbed into the cells and concentrated in perinuclear organelles by a non-receptor mediated process. The BODIPY derivative turned out to be chemically unstable and was bleached by the laser beam very rapidly. Rhodamine Green-heptagastrin retained a high affinity toward the gastrin receptor (Kd--45 nm in displacement of 125I-labeled cholecystokinin-8) and showed specific binding to NIH/3T3 cells stably transfected with human gastrin/CCK B receptor cDNA, but not to nontransfected 3T3 cells. The fluorescent signal of all three dyes was sufficiently intense for localization of the compounds in cells by means of CLSM. Rhodamine Green derivative was found to be a useful tool for the study of endocytosis of the hormone. It can also be utilized for quantitative estimation of binding and determination of Kd instead of the traditionally used radiolabeled derivatives of gastrin.

Visualization of G Protein-coupled Receptor Trafficking with the Aid of the Green Fluorescent Protein

“…In a previous study we compared the properties of three fluorescent derivatives of gastrin, rhodamine green, BODIPY, and Cy3.29 heptagastrin (25). The rhodamine green (RG) moiety provided the strongest fluorescence intensity, chemical and light stability and resistance to intracellular degradation within the cells necessary for the kinetic studies of ligand uptake.…”

“…Cy3.29 derivatives have emission maximum at 570 nm and do not emit below 540 nm and thus allow for simultaneous detection of the GFP fluorescence. Rhodamine green and Cy3.29-CCK-8 were obtained by the method used earlier for the synthesis of heptagastrin derivatives (25). The structures of the compounds were confirmed by NMR and mass spectroscopy.…”

Endocytosis of gastrin in cancer cells expressing gastrin/CCK-B receptor

“…The mechanisms of endocytosis of GRCP were studied for a small number of receptors (Ghinea et al 1992;Hoxie et al 1993;Ashworth et al 1995;Grady et al 1995;Roettger et al 1995) but the data are incomplete and controversial. We have previously described the synthesis and characterization of three fluorescent derivatives of heptagastrin (Czerwinski et al 1995). One of the derivatives, rhodamine green heptagastrin (RG-7G), was found to interact specifically with the gastrin/CCK-B receptor while retaining the biological properties of the parent peptide.…”

Cytotoxic agents directed to peptide hormone receptors: Defining the requirements for a successful drug

“…We estimated that K d for the fluorescent gastrin derivatives was in the nanomolar range (e.g., 2 nM for rhodamine green-7G conjugate; ref. 28). Therefore, we would predict that the resultant cytotoxic effect of a drug conjugated with a peptide ligand would become apparent only close to the concentration defined by K d .…”

“…Subsequent studies in our laboratory, which used fluorescent-labeled heptagastrin and CCK-8 (11,28,29), showed that both CCKBR and CCK-A receptor agonists undergo internalization by endocytosis upon binding to the receptors. The receptors and ligands are sorted in endosomes (11).…”