Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.

Abstract

Highly fluorescent analogs of insulin and e idermal growth factor were repared by the covalent attachment of these peptides to a--actalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The a-lactalbumin was specifically linked to the lysine residue of insulin or to the a-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8 Fluorescence microscopy is used extensively for localizing different cell surface components such as surface antigens (1), lectin-binding sites (2), and toxin receptors (3). Recently, the fluorescent photobleaching recovery technique has made it possible to study quantitatively the dynamic properties of such fluorescently labeled molecules on viable cells under physiological conditions (4, 5).Unfortunately, in only a few cases has it been possible to label hormone receptors with fluorescent analogs (6, 7). The number of hormone receptor sites per cell is generally so small (i.e., 10,000 to 40,000, at most) that a density of labeled receptors sufficiently high for microscopic detection is in theory difficult to achieve. Furthermore, polypeptide hormones have few potential sites for substitution with fluorophores without impairing the binding affinity of the hormone to its receptor. This makes it difficult or impossible to achieve by conventional methods the high fluorescence intensity that can be obtained by multiple fluorescent probe substitution. Extensive chemical modification of hormones may also cause gross changes in conformation, resulting in lowered affinity, specificity, and selectivity of binding to receptors on intact cells.In the present studies these problems are overcome by coupling to peptide hormones a protein (a-lactalbumin) that has previously been highly substituted (i.e., seven to one) with rhodamine through single, uniquely specified hormone residues whose selective modification is known not to adversely alter the biological activity of the hormone. Thus, a highly fluorescent analog of a-lactalbumin can be coupled to the a-amino group of epidermal growth factor (EGF) or to the e-amino group of the single lysyl residue of insulin. These derivatives retain partial biological activity, and they label receptors sufficiently well to permit localization by fluorescent microscopy and quantitation of lateral mobility by fluorescent photobleaching recovery methods.MATERIALS AND METHODS Materials. a-Lactalbumin, 5,5'-dithiobis(2-nitrobenzoic acid), dithiothreitol, iodoacetamide, 2-iodoacetic acid, citraconic anhydride, dansyl chloride, and S-acetylmercaptosuccinic anhydride were purchased from Sigma. Rhodamine (Rhod) isothiocyanate (tetramethyl form, hereafter referred to simply as rhodamine) was obtained from Research Organic Chemicals, 2,4-dinitrofluorobenzene and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MIB-NHS) from Pierce Products, and pork insulin from Eli'Lilly. EGF was purified from male mouse submaxillary glands ac...

Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

“…were found for these mutants, whether or not they were cleaved with CNBr. Experiments on EGF and TGF-a have shown that an N-terminal extension does not markedly modify EGF-binding activity (12,26). Therefore, the loss of activity obtained with [Met-48]-TGF-a that has not been CNBr treated was probably due to the mutation itself and not to the N-terminally extended fusion protein.…”

Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility.

“…In addition to being phosphorylated, insulin-occupied receptors move laterally within the plasma membrane and into clathrin-coated pits (25,31,(34)(35)(36)(37). Thus in the presence ofinsulin, there is a net flux ofoccupied receptors into coated pits, from which they enter the coated vesicle-endosome continuum.…”

Nuclear Targeting of Chlorin e6 Enhances Its Photosensitizing Activity

“…Chlorin e 6 was initially reacted with 1,6-diaminohexane (Sigma) using CMCS (ratio of chlorin e 6 to 1,6-diaminohexane to CMCS of 1:100:100) in 10 mM sodium phosphate buffer, pH 7.0, and CMCS was subsequently used to link aminochlorin e 6 with the P10/␤-galactosidase carrier in the same buffer (ratio of carrier to aminochlorin e 6 to CMCS of 1:30:300). After successive reaction of insulin with citraconic anhydride (18) (Serva) and SPDP (ratio of 1:6) in 25 mM Hepes, 150 mM NaCl, pH 7.5 (buffer B), citraconic-insulin-PDP was linked to P10-chlorin e 6 or ␤-galactosidase-chlorin e 6 (ratio of 15:1) in buffer B, after which the citraconic groups were removed (18). Two series of constructs were made: P10-(chlorin e 6 )-insulin (1:5:7), ␤-galactosidase-(chlorin e 6 )-insulin (1:5: 7), and P10-(chlorin e 6 )-insulin (1:3:8) and ␤-galactosidase-(chlorin e 6 )-insulin (1:2:8).…”