Iodoazidobenzylpindolol, a photoaffinity probe for the beta-adrenergic receptor.

Abstract

A high-affinity pindolol derivative, (±)-1-(indol-4-yloxy)-3-[1-(p-azido-m-iodophenyl)-2-isobutylamine]-2-propanol (IABP), has been prepared; it contains an iodide and an azide functional group and acts as a photoaffinity label for the f-adrenergic receptor. When [125I]IABP (specific activity 1300 Ci/ mmol) was photolyzed with crude duck erythrocyte membrane preparations, which contain 3-adrenergic receptor binding sites, highly specific labeling of two polypeptides was observed upon electrophoresis on sodium dodecyl s ate/polyacrylamide gels. The -adrenergic receptor is a protein (1) located in the plasma membrane of various cultured cell lines (2-6), nucleated erythrocytes (7-9), and several mammalian tissues (cardiac muscle, bronchial smooth muscle) that respond to epinephrine. This receptor is functionally coupled to the enzyme adenylate cyclase via the nucleotide regulatory protein (10). Many of the ligand binding properties of this receptor have been studied by utilizing radioactive (3-receptor antagonists (7, 11, 12) and agonists (13,14). One of the competitive antagonists that has demonstrated very high affinity for the ( receptor and has been prepared at the highest specific radioactivity is iodohydroxybenzylpindolol (11, 15, 16). In order to identify the polypeptide or polypeptides that make up this catecholamine receptor, suitable derivatives of ,B receptor antagonists must be available for forming covalent bonds in the binding site. This method for specifically "tagging" a binding site in heterogeneous mixture of binding sites by utilizing chemically reactive site-directed ligands has been described as "affinity labeling" (17)(18)(19); more recently the method of "photoaffinity labeling" has been used (20-23). The use of light-activated compounds possesses several inherent advantages over regular affinity labeling for increasing the specificity of the derivatization reaction (24). Some affinity and photoaffinity labels for the (3receptor have been reported. A bromoacetyl derivative of propranolol (25) and a bromoacetyl derivative of alprenolol (26) were reported as specific irreversible inhibitors of the (3adrenergic receptor.

125I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage.

“…Photolysis of iodinated organic compounds with 254-nm light has been shown to cleave carbon-iodine bonds yielding carbon and iodine radicals (28), but this clearly did not occur under our conditions using 366-nm light, which is in agreement with previous studies (19,(21)(22)(23)(24). No radioactive proteins were present in the Sepharose eluate after photolysis alone (Fig.…”

“…In addition, the radioactive labeling that occurs following photolysis with 366-nm light is due to reactions mediated by the aryl nitrene and is not due to the incorporation of free 125I. This latter result is in agreement with those of others (19,(21)(22)(23)(24). …”

Effects of pressure overload, left ventricular hypertrophy on beta-adrenergic receptors, and responsiveness to catecholamines.

“…The future development ofmethods that permit both purification (17,18) and photoaffinity labeling ofthe hormone-binding subunit (17)(18)(19)(20) will permit this hypothesis to be examined at the molecular level.…”

Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain.

“…Characterization of the BAR has been aided by the development of radioligand binding techniques and, most particularly, by the availability of irreversible photoaffinity ligands (4)(5)(6); the use of such specific probes allows determination of the properties of the protein without prior purification to homogeneity. The small size of adrenergic ligands limits their usefulness in localization studies, however, as they cannot easily accommodate fluorescent or electron-dense markers.…”