Ligand Specificity and Conformational Dependence of the Hepatic Nuclear Factor-4α (HNF-4α)

Abstract

Hepatic nuclear factor-4␣ (HNF-4␣ Hepatic nuclear factor 4 (HNF-4)1 is a member of the superfamily of nuclear receptors which includes steroid hormone receptors and nonsteroid ligand dependent transcription factors, e.g. thyroid hormone receptor, retinoid X receptor, retinoid acid receptor, and peroxisome proliferator-activated receptors (PPARs) (reviewed in Ref. 1). HNF-4␣ isoforms (␣ 1 -␣ 3 ) have been cloned and characterized and are expressed in mammals in liver, kidney, intestine, and pancreas (reviewed in Ref.2). Unlike retinoid X receptor ␣, with which it has 40% amino acid sequence homology, HNF-4 does not form heterodimers with any other nuclear receptor, but binds to direct repeat-1 DNA sequences as homodimer (3). Direct repeat-1 motifs are promiscuous binding sites for HNF-4, PPAR, retinoid acid receptor, retinoid X receptor, chicken ovalbumin upstream-promoter transcription factor, and chicken ovalbumin upstream-promoter transcription factor homo-or heterodimers (4).HNF-4␣ responsive genes encode transcription factors (HNF-1␣, PXR), proteins involved in fatty acid, lipoprotein, and lipid metabolism (apoA-I, apoA-II, apoB, apoC-II, apoC-III, L p (a), microsomal triglyceride transfer protein, mitochondrial fatty acyl-CoA dehydrogenases, and fatty acid-binding protein), carbohydrate metabolism (insulin, glut2, glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, pyruvate kinase, aldolase B, and glyceraldehyde-3-phosphate dehydrogenase), amino acid and protein metabolism (ornithine transcarbamylase, tyrosine aminotransferase, phenylalanine hydroxylase, and antitrypsin ␣ 1 ), P450 enzymes (steroid 15␣-hydroxylase, fatty acyl -hydroxylase, cholesterol-7␣-hydroxylase, and drug metabolizing P450 enzymes (cyp3␣ 4 -6)), hematopoiesis (erythropoietin and transferrin), blood coagulation (factors VII, IX, and X and fibrinogen), and others (e.g. cellular retinolbinding protein and transthyretin) (reviewed in Refs. 2 and 5-14). Since HNF-4␣ activates the transcription of some nuclear receptors (HNF-1␣) and may further directly interact with other transcription factors (HNF-1␣), the above list of HNF-4␣ responsive genes may include some which are transcriptionally affected by HNF-4␣ indirectly.The first demonstration of putative HNF-4 ligands showed that long chain fatty acyl-CoA (LCFA-CoAs) thioesters (15), as well as CoA-thioesters of hypolipidemic peroxisome prolifera-

Very-Long-Chain and Branched-Chain Fatty Acyl-CoAs Are High Affinity Ligands for the Peroxisome Proliferator-Activated Receptor α (PPARα)

“…The direct binding of non-fluorescent ligands to PPARαΔAB was carried out as described (30,35). Briefly, PPARαΔAB (0.1μM in 2ml of PBS, pH 7.4) was titrated with increasing ligand (0-2000nM).…”

“…Circular dichroic spectra of PPARαΔAB (0.8μM in 125μM HEPES, pH 8.0, 12.5μM DTT, 5mM KCl, 0.3% glycerol) were taken in the presence and absence of ligands (20μM) with a J-710 spectropolarimeter (JASCO Inc., Easton, MD) at 23°C in a 1-mm cuvette as described (30,35). Spectra were recorded from 280 to 185nm with a bandwidth of 2nm, sensitivity of 10 millidegrees, scan rate of 50nm/min, and a time constant of 1s.…”

“…in (18,19), an assay not dependent upon an exogenous radiolabeled or fluorescent ligand or upon physical separation of bound from free ligand was utilized. This assay, recently developed by our laboratory (30,35), takes advantage of PPARα's intrinsic aromatic amino acid fluorescence which is quenched upon ligand binding. In the case of saturated VLCFA, saturation binding curves were obtained for the 20-carbon arachidic acid (Fig.…”

“…Ligand-induced conformational change is a hallmark of ligand-regulated nuclear transcription factors, including PPAR (34,35,41,43). The observation that binding of non-fluorescent ligands significantly quenched the emission of PPARαΔAB aromatic amino acid residues (Figs.…”

Role of Regulatory F-domain in Hepatocyte Nuclear Factor-4α Ligand Specificity

“…1) (8,(11)(12)(13)(14)(15)(16). The molecular basis for this constitutive activity remains to be determined but may include ligand-based mechanisms involving dietary or endogenous long chain fatty acids (LCFA) (17,18), endogenously synthesized coenzyme A (CoA) thioesters of long chain fatty acids (LCFA-CoAs) (8,11,14,19), and/or the F-domain itself functioning as a modulator (16).…”