Visualization of multiple imidazoline/guanidinium-receptive sites

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Compounds with an imidazoline or guanidinium moiety elicit a variety of stimulatory and inhibitory cell responses in both central and peripheral tissues. Many of these effects are mediated by interaction with alpha-adrenergic receptors, but these molecules also selectively recognize other membrane-bound proteins with high affinity. We used a functionalized derivative of the imidazoline molecule cirazoline to visualize the imidazoline/guanidinium-receptive site (IGRS). 2-[3-Aminophenoxy]methyl imidazoline was radioiodinated and subsequently converted to the arylazide to generate the photoaffinity adduct 2-[3-azido-4-[125I]iodophenoxy]methyl imidazoline ([125I]AZIPI). Both 2-[3-amino-4-[125I]iodophenoxy]methyl imidazoline and [125I]AZIPI exhibited saturable high affinity binding in rat liver membrane preparations (Ki = 2-5 nM). In rat liver mitochondrial membranes, [125I]AZIPI photoincorporates into two peptides with apparent molecular weights of approximately 55,000 and approximately 61,000 as determined by SDS-polyacrylamide gel electrophoresis. The labeling of these two species is blocked by various competing ligands (10 microM) with a potency order expected for an IGRS. The photolabeling of both peptides is blocked by the imidazolines cirazoline and idazoxan or by the guanidinium guanabenz, but it is not altered by the alpha 2-adrenergic receptor antagonist rauwolscine or by the adrenergic receptor agonist epinephrine. Photoincorporation of [125I]AZIPI is minimally inhibited by the imidazoline clonidine or by the alpha 1-adrenergic receptor antagonist prazosin. However, the guanidinium ligand amiloride exhibits higher affinity for the M(r) = 61,000 peptide as compared with the M(r) = 55,000 peptide, suggesting that the two labeled species differ in their ligand recognition properties. An additional IGRS was identified by photolabeling in membranes prepared from PC-12 pheochromocytoma cells. In PC-12 membranes, [125I]AZIPI photolabels a major M(r) = approximately 61,000 peptide; the photoincorporation is blocked by cirazoline, guanabenz, and amiloride but not by idazoxan (competing ligands = 10 microM). These data indicate the existence of at least three subtypes of IGRS that differ in their ligand recognition properties, their apparent molecular weight, and their tissue distribution.