CAR: Three new models for a problem child

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Table 1. Adiposity and insulin resistance: Not always a simple correlation

Adipose triacylglycerol stores Adipocyte number Insulin resistance Liver triacylglycerol stores

Obese

Lean

Lipodystrophy

fld

+ Adipose lipin

+ Muscle lipin

up

down

down

down

up

up

up

down or no change down down

down

?

?

?

up up

up biphasic

down ?

up ?

up up

Note the difference from the usual correlation between adipose lipid content and insulin resistance and liver lipid content in lipodystrophy, the fld mouse, and the adipose lipin mouse but not in the muscle lipin mouse. Data are from Phan and Reue (2005), Reitman (2002), and references therein.

Two closely related genes, Lpin2 and Lpin3, are present in mice and humans (Peterfy et al., 2001). To date, no characterization has been reported for them. LPIN2 and LPIN3 show a broad tissue distribution in transcript databases, but it is not known if they complement Lpin1 function or if they are targets of insulin signaling. One possibility is that the observed fld phenotype is determined by the tissues that lack all lipin-like proteins (loss of Lpin1 coupled with normal lack of Lpin2 and Lpin3 expression). Lipodystrophy and leanness are quite distinct conditions (Table 1). One way to conceptualize the difference is that, in lipodystrophy, there is a deficiency of adipose tissue (both cells and lipid cargo), while, in leanness, the adipose tissue is present, but its triacylglycerol content is reduced. In lipodystrophy, as a result of the missing adipocytes, there is increased triacylglycerol deposition in nonadipose tissues, particularly liver. Such lipotoxicity is a correlate of tissue insulin resis-

tance. The contrasting physiology of lipodystrophy and leanness may explain the clinical observation that weight loss of 10 kg by liposuction does not improve insulin sensitivity, whereas this degree of weight loss by diet and exercise produces significant metabolic benefit (Klein et al., 2004). The Phan and Reue experiments (Phan and Reue, 2005) showing obesity as a consequence of lipin overexpression complement the lipodystrophy resulting from lipin absence. Unanswered questions about lipin include the details of its molecular function, how its overexpression causes the observed phenotypes, why hepatic steatosis is present only transiently in the fld mouse, and why the fld mouse develops peripheral neuropathy. Presumably, this information will mesh with and complement information derived from other models of lipodystrophy and of obesity. Clearly, the lipin field has more mystery and fun ahead.

Marc L. Reitman Metabolic Disorders Merck Research Laboratories Rahway, New Jersey 07065

Selected reading Franckhauser, S., Munoz, S., Pujol, A., Casellas, A., Riu, E., Otaegui, P., Su, B., and Bosch, F. (2002). Diabetes 51, 624–630. Garg, A. (2004). N. Engl. J. Med. 350, 1220– 1234. Gavrilova, O., Marcus-Samuels, B., Graham, D., Kim, J.K., Shulman, G.I., Castle, A.L., Vinson, C., Eckhaus, M., and Reitman, M.L. (2000). J. Clin. Invest. 105, 271–278. Hotamisligil, G.S., Johnson, R.S., Distel, R.J., Ellis, R., Papaioannou, V.E., and Spiegelman, B.M. (1996). Science 274, 1377–1379. Huffman, T.A., Mothe-Satney, I., and Lawrence, J.C. (2002). Proc. Natl. Acad. Sci. USA 99, 1047–1052. Klein, S., Fontana, L., Young, V.L., Coggan, A.R., Kilo, C., Patterson, B.W., and Mohammed, B.S. (2004). N. Engl. J. Med. 350, 2549–2557. Peterfy, M., Phan, J., Xu, P., and Reue, K. (2001). Nat. Genet. 27, 121–124. Phan, J., and Reue, K. (2005). Cell Metabolism 1, this issue, 73–83. Phan, J., Peterfy, M., and Reue, K. (2004). J. Biol. Chem. 279, 29558–29564. Reitman, M.L. (2002). Annu. Rev. Nutr. 22, 459– 482. Shepherd, P.R., Gnudi, L., Tozzo, E., Yang, H., Leach, F., and Kahn, B.B. (1993). J. Biol. Chem. 268, 22243–22246. Shimomura, I., Hammer, R.E., Ikemoto, S., Brown, M.S., and Goldstein, J.L. (1999). Nature 401, 73–76. DOI 10.1016/j.cmet.2004.12.005

CAR: Three new models for a problem child By comparison with its older and better-behaved cousins in the nuclear receptor superfamily, CAR (NR1I3) has always been an oddball. Three new crystal structures recently described in Molecular Cell reveal the molecular basis for some of its bad habits (Xu et al., 2004; Suino et al., 2004; Shan et al., 2004). CAR’s unruly behavior began with a difficult birth. Its initial description (Baes et al., 1994) was carefully considered and rejected by three top journals skeptical of its function as a constitutive transcriptional activator, behavior that is now known to be common among or-

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phan receptors but was considered deviant at the time. And it really was an orphan when it emerged first as MB67, with its original name (constitutive activator of retinoid response) a casualty of the peer review battle. CAR (now constitutive androstane re-

ceptor) regained its birthright with the identification of its first ligands, androstanol and androstenol (Forman et al., 1998). But conventional wisdom was challenged again, since these ligands deactivated rather than activated CAR. After further struggles with more descrip-

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tive alternatives, the journal insisted on “inverse agonists,” a pharmacologic term that has confused molecular biologists ever since. (While conventional antagonists block the function of agonists, inverse agonists block basal activity of their target.) The first real clue to the physiologic role of CAR came with the suggestion that it was involved in the induction of cytochrome P450 gene expression by a series of drugs and foreign compounds exemplified by phenobarbital (PB) (Honkakoski et al., 1998). This was emphatically borne out by the complete loss of induction of drug metabolism in response to appropriate xenobiotics in a CAR knockout mouse (Wei et al., 2000). CAR thus joined its closest relative PXR as a “xenosensor.” Drug metabolism is a central aspect of liver function with obvious and important clinical implications. By inducing drug clearance pathways in response to a very wide range of xenobiotics, CAR and PXR generally function to protect us from exogenous chemical threats. Several recent studies suggest that CAR also controls metabolism of endobiotics, including bilirubin (Huang et al., 2003) and bile acids (Saini et al., 2004; Zhang et al., 2004), endogenous toxins, as well as key metabolic regulators such as thyroid hormone (Maglich et al., 2004; Qatanani et al., 2004). The chemical defense response controlled by CAR and PXR is reminiscent of the immune system in its ability to respond to a virtually unlimited number of foreign compounds. However, it is based on an essentially opposite strategy. Thus, both receptors respond to a wide range of structurally diverse compounds rather than to a unique hormone, and their cytochrome P450 targets and other downstream effectors have similarly extended substrate specificities. This iconoclastic behavior adds to the list of questions regarding the unusual functions of CAR. What is the molecular basis for constitutive transactivation? How do inverse agonists work? How does it respond to diverse xenobiotics? The new structures provide a complex but coherent answer to the question of constitutive activity. In general, the liganddependent coactivator binding surface of the nuclear receptors, termed AF-2, is dependent on appropriate positioning of the C-terminal helix 12. Agonist ligands often make direct contact with the inner

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Figure 1. A schematic model depicting the active-to-inactive state transition of CAR In the crystal structures of the active form of CAR, helix 12 of apo-CAR is stabilized in the active conformation by the constraints of the short linker helix HX and hydrogen bonds (arrows) between the C-terminal carboxylate and residues K205 from helix H4 and S337 from helix H10 (Xu et al., 2004; Suino et al., 2004). This allows binding of coactivators via their conserved LXXLL motifs. Binding of the inverse agonist androstenol to CAR promotes a collapse of the apo-pocket by introducing a kink of helix 10 at S337, which results in displacement of the linker helix and AF-2 from the active configuration and dissociation of coactivator LXXLL motifs (Shan et al., 2004) (E. Xu, personal communication).

surface of this conformationally flexible helix to hold it in place and also stabilize appropriate positions of other nearby helices. As expected, helix 12 adopts the active conformation in the structures of human CAR bound to its agonists, CITCO and 5β-pregananedione, but folds across a barrier of four internal amino acids rather than capping the ligand binding pocket (Xu et al., 2004). Two conserved features unique to CAR promote the active conformation of helix 12. The flexible loop that connects it to helix 10 in other receptors is replaced by a short helix that holds it in place more rigidly (Figure 1). At the other end, the lack of the usual C-terminal sequence extension allows the terminal carboxyl group to form a previously predicted (Dussault et al., 2002) hydrogen bond with a conserved lysine in helix 4 and also with S337 from helix H10 (Figure 1). In essence, the helix is fastened to the rest of the LBD like a staple. This model for the molecular basis of constitutive activity contrasts strongly with others. In the Nurr1 structure, for instance, bulky side chains fill the ligand pocket and stabilize the active conformation (Wang et al., 2003). An extended helix 2 stabilizes the active LRH-1 structure, despite an empty pocket (Sablin et al., 2003). The new structures suggest

that helix 2 reinforcement and possibly also the large surface of heterodimerization with RXR may also stabilize the active conformation of apo-CAR. In contrast to the human structures, the potent mouse CAR agonist TCPOBOP pokes through the barrier and directly contacts both helix 12 and the linker helix (Suino et al., 2004). This presumably explains its ability to superactivate murine CAR in transfection studies. The presence of the barrier also neatly explains the lack of such a response of human CAR to CITCO in similar studies but raises the question of the basis for the potent biologic effects of CITCO. The answer to this question lies in another unique CAR activity. In the liver but not in cultured cell lines, CAR is normally sequestered in the cytoplasm. It translocates to the nucleus in response to a poorly understood signal elicited by a range of secondary activators, including PB, bilirubin, and bile acids, which do not bind CAR (Swales and Negishi, 2004). In such cases, the activation of appropriate target genes is clearly dependent on the constitutive activity, but translocation can also be induced by direct ligands like CITCO. But if ligand binding is a dispensable aspect of CAR’s primary xenosensor function, why does it have a pocket at

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all? The structures of the TCPOBOP and androstanol bound murine CAR show that the pocket allows not only translocation in response to specific xenobiotics but also more conventional allosteric modulation of CAR activity. Interestingly, the mechanism of the inhibitory effects of the inverse agonist differs from that of previously described nuclear receptor antagonists, which typically include a moiety that projects into the space occupied by helix 12 and actively disrupts the AF-2 surface. By contrast, androstenol literally undermines the mechanisms that promote the constitutive activity of CAR (Figure 1). In the androstenol bound structure (Shan et al., 2004), the additional helix that stabilizes helix 12 is displaced inward toward the ligand binding cavity. This realigns the C-terminal helix and breaks the hydrogen bond that tacks it down to helix 4. Coactivators can no longer bind, but corepressors may. This mechanism is reminiscent of the “passive” antagonism in which THC binding to ERβ subtly alters the conformation of helix 10 (in the nomenclature used for CAR) and thereby stabilizes an inactive helix 12 conformation (Nettles et al., 2004). Modulation of the positioning of the C terminus of helix 10, which includes the dimer interface, has been suggested to be a key aspect of the allosteric effects of both ligand binding and dimerization (Nettles et al., 2004). While the new structures can explain some of CAR’s odd habits, they also raise a number of questions. One of particular interest is the role of RXR. At least in the human CAR complexes, there is a direct contact between CAR helix 7 and the C terminus of RXR. This appears to be dependent on the active conformation of RXR, which is unexpectedly promoted by an adventitiously bound fatty

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acid in the RXR ligand pocket. Predictably, perhaps, RXR agonists can have unusual effects on CAR (Tzameli et al., 2003). Thus, the RXR partner may have a more dynamic role than suggested by the necessarily static crystal structures. More broadly, these structures provide a firm foundation for design of new pharmacologic tools to modulate CAR activity. CAR’s function in drug metabolism suggests that such compounds could either promote the clearance of toxic xenobiotics or prevent the formation of toxic metabolites. However, if we have learned anything about CAR in the last ten years, it is to expect the unexpected, and functions in processes beyond drug metabolism are emerging. Thus, it has recently been suggested as a therapeutic target for jaundice (Huang et al., 2004). Based on its potential to modulate thyroid hormone levels, inhibition of CAR has also been suggested to have beneficial effects on weight loss (Maglich et al., 2004). Who knows where it will lead us next? David D. Moore Department of Molecular and Cellular Biology Baylor College of Medicine 1 Baylor Plaza Houston, Texas 77030

Huang, W., Zhang, J., Chua, S.S., Qatanani, M., Han, Y., Granata, R., and Moore, D.D. (2003). Proc. Natl. Acad. Sci. USA 100, 4156–4161. Huang, W., Zhang, J., and Moore, D.D. (2004). J. Clin. Invest. 113, 137–143. Maglich, J.M., Watson, J., McMillen, P.J., Goodwin, B., Willson, T.M., and Moore, J.T. (2004). J. Biol. Chem. 279, 19832–19838. Nettles, K.W., Sun, J., Radek, J.T., Sheng, S., Rodriguez, A.L., Katzenellenbogen, J.A., Katzenellenbogen, B.S., and Greene, G.L. (2004). Mol. Cell 13, 317–327. Qatanani, M., Zhang, J., and Moore, D.D. (2004). Endocrinology 13, in press. Published online November 24, 2004. 10.1210/en.2004-1350. Sablin, E.P., Krylova, I.N., Fletterick, R.J., and Ingraham, H.A. (2003). Mol. Cell 11, 1575–1585. Saini, S.P., Sonoda, J., Xu, L., Toma, D., Uppal, H., Mu, Y., Ren, S., Moore, D.D., Evans, R.M., and Xie, W. (2004). Mol. Pharmacol. 65, 292– 300. Shan, L., Vincent, J., Brunzelle, J.S., Dussault, I., Lin, M., Ianculescu, I., Sherman, M.A., Forman, B.M., and Fernandez, E.J. (2004). Mol. Cell 16, 907–917. Suino, K., Peng, L., Reynolds, R., Li, Y., Cha, J.Y., Repa, J.J., Kliewer, S.A., and Xu, H.E. (2004). Mol. Cell 16, 893–905. Swales, K., and Negishi, M. (2004). Mol. Endocrinol. 18, 1589–1598. Tzameli, I., Chua, S.S., Cheskis, B., and Moore, D.D. (2003). Nucl. Recept. 1, 2.

Selected reading

Wang, Z., Benoit, G., Liu, J., Prasad, S., Aarnisalo, P., Liu, X., Xu, H., Walker, N.P., and Perlmann, T. (2003). Nature 423, 555–560.

Baes, M., Gulick, T., Choi, H.-S., Martinoli, M.G., Simha, D., and Moore, D.D. (1994). Mol. Cell. Biol. 14, 1544–1552.

Wei, P., Zhang, J., Egan-Hafley, M., Liang, S., and Moore, D.D. (2000). Nature 407, 920–923.

Dussault, I., Lin, M., Hollister, K., Fan, M., Termini, J., Sherman, M.A., and Forman, B.M. (2002). Mol. Cell. Biol. 22, 5270–5280.

Xu, R.X., Lambert, M.H., Wisely, B.B., Warren, E.N., Weinert, E.E., Waitt, G.M., Williams, J.D., Collins, J.L., Moore, L.B., Willson, T.M., and Moore, J.T. (2004). Mol. Cell 16, 919–928.

Forman, B.M., Tzameli, I., Choi, H.S., Chen, J., Simha, D., Seol, W., Evans, R.M., and Moore, D.D. (1998). Nature 395, 612–615. Honkakoski, P., Zelko, I., Sueyoshi, T., and Negishi, M. (1998). Mol. Cell. Biol. 18, 5652–5658.

Zhang, J., Huang, W., Qatanani, M., Evans, R.M., and Moore, D.D. (2004). J. Biol. Chem. 279, 49517–49522. DOI 10.1016/j.cmet.2004.12.006

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