Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXR␤ as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXR␤ is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXR␣ and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXR␤. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXR␤ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.Cholesterol metabolism is a tightly regulated enzymatic pathway. Deregulation of this pathway leads to accumulation of excess cholesterol and can result in diseases such as atherosclerosis and gallstone formation (10). The homeostatic balance between uptake and elimination of cholesterol is accomplished by regulation of three pathways: de novo cholesterol synthesis from acetate, uptake of cholesterol from the intestine, and elimination of cholesterol through the synthesis of bile acids.Cholesterol catabolism into bile acids is controlled by transcriptional feedback and feedforward mechanisms that are mediated by members of the nuclear receptor family. Activation of cholesterol breakdown into bile acids is mediated by liver X receptor alpha (LXR␣; NR1H3), a nuclear receptor that binds oxysterols formed during the synthesis and metabolism of cholesterol (14,18,28). Together with another nuclear receptor, liver receptor homologue 1 (NR5A2), LXR␣ stimulates transcription of cholesterol 7␣-hydroxylase (CYP7A1), an enzyme catalyzing the rate-limiting step of this pathway. Suppression of cholesterol degradation into bile acids is triggered by the farnesoid X receptor (FXR; NR1H4), which binds to and is activated by bile acids (21, 27, 33). Ligand-bound FXR activates transcription of the short heterodimer partner (NR0B2) and subsequently downregulates transcription of CYP7A1 (11, 19). However, there are species differences in the regulation of cholesterol metabolism and sensitivity to dietary cholesterol. While rodents respond to cholesterol feeding with induction of CYP7A1, humans and rabbits appear to lack this response and are left more sensitive to the cholesterolemic effects of dietary cholesterol (13,35,36). This difference was directly attributed to the ability of LXR to regulate CYP7A in different species (4, 24).Nuclear hormone receptors form a famil...

Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideum myosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K A ) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K A stem almost exclusively from variations in the dissociation rate constant (k -A ), with the introduction of a single negative charge at position 532 having the same effect on k -A as the introduction of 12 positive charges in the loop 2 region.Myosin II drives muscle contraction and motile processes such as cytokinesis and cell motility. The ATP-dependent 1 interaction of the myosin head with actin is central to myosin driven motile processes. Atomic models of the actomyosin rigor complex show the interface between myosin and actin to consist of four major contact regions, suggesting a sequential mechanism of binding (1, 2). A first contact involves a highly charged, lysine-rich loop on myosin (loop 2) formed by residues S619 to V630 in the case of Dictyostelium discoideum myosin II and a cluster of acidic residues at the N-terminus of actin (3-6). A second contact region involves a helix-loop-helix structure formed by residues S510 to K546 (unless otherwise stated amino acid residues are numbered according to the D. discoideum myosin II sequence). A loop formed by residues L399 to V411 makes a third contact. These three contacts involve a single actin monomer and form the primary actin-binding site of myosin. In addition a loop protruding between residues L547 and H572 may reach the neighboring actin monomer one actin helix turn below (Figure 1).Previously, we have shown that the light-chain-binding domain (LCBD) plays no major role in the biochemical behavior of the myosin (7-9). Recombinant constructs without LCBD can be produced and purified in large amounts and are ideally suited for systematic studies of the structure, kinetics and function of the myosin motor. Therefore, we used construct M765, corresponding to the first 765 residues of D. disc...