The F₁-ATPase β-subunit is the putative enterostatin receptor

Abstract

It has been suggested that the F₁-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin_1–7, in the absence and presence of cold enterostatin. ¹²⁵I-β-casomorphin_1–7 weakly binds to the rat F₁-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin_1–7 binding to the F₁-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin_1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin_1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F₁-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F₁-ATPase complex. Western blot analysis showed the F₁-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F₁-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin_1–7 bind to distinct sites on the protein.

Introduction

Enterostatin (APGPR) is the aminoterminal pentapeptide of procolipase and is known to induce satiation in rats [21]. Specifically, it suppresses intake of a high fat diet, but not a high carbohydrate diet when administrated centrally or peripherally to overnight fasted rats [3], [6], [14]. Enterostatin is released from pancreatic procolipase by proteolytic activity in the small intestine [4]. Procolipase and enterostatin have also been localized to the gastric mucosa and to certain brain regions suggesting that the gene is expressed in both the gastrointestinal tract and the central nervous system [11], [15]. Enterostatin (38 nmol) inhibited, while β-casomorphin at the same dose stimulated the intake of high fat diet in the rat [7], [20]. This observation, together with the similarity of the amino acid sequences, suggested an agonist–antagonist relationship for the two peptides. At a higher dose, enterostatin also increased intake of a high fat diet [6], possibly reflecting activity as a partial antagonist or suggesting that enterostatin and β-casomorphin have separate binding sites on a receptor.

To understand the regulation of fat intake by enterostatin, the identification of a receptor or binding protein for enterostatin is crucial. Erlanson-Albertsson and coworkers previously reported the mitochondrial F₁-ATPase β-subunit as a potential receptor for enterostatin [2] by purifying F₁-ATPase β-subunit from rat brain with enterostatin affinity chromatography and showing binding of a labeled enterostatin analog YGGAPGPR with purified bovine F₁-ATPase β-subunit. They also showed that β-casomorphin_1–5 (YPFPG) competed with the peptide for binding. Membrane-binding studies have also suggested the presence of both high- and low-affinity sites for enterostatin [1]. The work reported in this paper provides evidence that the F₁-ATPase β-subunit is the receptor for enterostatin.