Background
Brigham Young University (1984)Ph.D., Purdue University (1990)Postdoctoral Fellow, University of California, Los Alamos National Lab (1990-92)Staff Scientist, University of California, Los Alamos National Lab (1992-96)
Cancer Research
Mechanisms of Assembly of Signaling Complexes: Most cellular functions are performed by proteins associated together into complexes. In fact, many proteins cannot even exist in the cell without their binding partners. These protein complexes often require the help of other proteins, called chaperones, to bring the complexes together. This is certainly the case for protein complexes involved in cell signaling processes. Our work has focused on one of these signaling complexes, the G protein heterotrimer. It is through the G protein complex and its associated receptors and effectors that cells detect hormones, neurotransmitters, chemokines and sensory signals such as odorants, taste molecules and even photons of light. G proteins regulate almost every aspect of cellular physiology and as a result more than a third of current therapeutic drugs target G protein signaling pathways.
In recent years, our lab has described the initial steps in the assembly of the G protein complex. This process begins with the association of the G protein β subunit (Gβ) with the G protein γ subunit (Gγ) into the Gβγ dimer. Gβγ is an obligate dimer, meaning that neither subunit is stable in the cell without the other. As a result, Gβ and Gγ must be brought together by chaperones. The way in which chaperone-mediated Gβγ assembly occurs is as follows. At some point during or after translation, the nascent Gβ subunit binds to the cytosolic chaperonin complex (CCT). CCT is a large protein-folding machine made up of a double-ring structure with eight different chaperonin subunits in each ring. Like other proteins folded by CCT, Gβ associates in the folding cavity in the center of the ring and is folded into a near-native conformation. However, Gβ cannot achieve its native fold and release from CCT without Gγ nor can Gβ associate with Gγ until it is released from CCT. This conundrum is resolved by the CCT co-chaperone, phosducin-like protein 1 (PhLP1). PhLP1 binds Gβ in the CCT folding cavity and initiates the release of Gβ from CCT. Once released, Gγ is able to bind Gβ in the PhLP1-Gβ complex and form the stable Gβγ dimer. The G protein α subunit then associates with Gβγ, forming the active Gαβγ heterotrimer and simultaneously releasing PhLP1. All four of the typical Gβ subunits are assembled with their 12 associated Gγ subunits by this same mechanism involving CCT and PhLP1.
The atypical Gβ5 subunit forms an obligate dimer with regulators of G protein signaling (RGS) proteins of the RGS7 subfamily. These dimers have a different function than Gβγ dimers. They turn off G protein signaling in neurons by accelerating the rate of GTP hydrolysis on the Gα subunit. We have found that CCT and PhLP1 also assist in the assembly of these Gβ5-RGS complexes. In fact, the conditional deletion of the PhLP1 gene in the rod photoreceptor cells of mice results in the loss of the Gβ5-RGS9 dimer from these cells in addition to the loss of Gβγ dimers. Consequently, G protein-dependent responses to light by rod photoreceptors were diminished and their recovery was slow. These findings have confirmed the importance of PhLP1 in Gβγ and Gβ5-RGS dimer formation in vivo.
Some of our more recent work has focused on the structural mechanism by which CCT and PhLP1 mediate Gβγ assembly. We have isolated two intermediates in the process of Gβγ assembly, the Gβ-CCT and the PhLP1-Gβ-CCT complexes and have determined their structures by cryo-EM in collaboration with the lab of Jose M. Valpuesta at the Centro National de Biotecnologia in Madrid, Spain. The cryo-EM structures show that Gβ folds into a near-native conformation within the CCT folding cavity and associates with the apical domain of the CCTγ subunit. PhLP1 binds Gβ from above the folding cavity, disrupting its interact with CCTγ and allowing PhLP1-Gβ to release from CCT for association with Gγ.
Publications
Lord, N. P., Plimpton, R. L., Sharkey, C. R., Suvorov, A., Lelito, J. P., Willardson, B. M., Bybee, S. M. (2016). A cure for the blues: opsin duplication and subfunctionalization for short-wavelength sensitivity in jewel beetles (Coleoptera: Buprestidae).. BMC evolutionary biology, 16(1), 107.
Xie, K., Masuho, I., Shih, C.-C., Cao, Y., Sasaki, K., Lai, C. W. J., Han, P.-L., Ueda, H., Dessauer, C. W., Ehrlich, M. E., Xu, B., Willardson, B. M., Martemyanov, K. A. (2015). Stable G protein-effector complexes in striatal neurons: mechanism of assembly and role in neurotransmitter signaling. eLife.
Plimpton, R. L.*, Cuéllar, J.*, Lai, C. W. J., Aoba, T., Makaju, A., Franklin, S., Mathis, A. D., Prince, J. T., Carrascosa, J. L., Valpuesta, J. M. and Willardson, B. M. (2015) “Structures of the Gβ‑CCT and PhLP1‑Gβ‑CCT Complexes Reveal a Molecular Mechanism for G protein β Subunit Folding and βγ Dimer Assembly”. Proc. Natl. Acad. Sci. U.S.A. 112, 2413-2418 (*Equal contribution of these two authors)
Tracy, C. M.*, Kolesnikov A. V.*, Blake D. R., Chen, C.-K., Baehr, W., Kefalov, V. J. and Willardson B. M. (2015) “Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.” PLOS ONE 10, e0117129 (*Equal contribution of these two authors)
Tracy, C. M., Gray A. J., Cuellar, J., Shaw, T.S., Howlett, A.C., Taylor, R.M., Prince, J.T., Ahn, N.G., Valpuesta, J.M. and Willardson, B.M. (2014) “Programmed cell death protein 5 interacts with the chaperonin CCT to regulate β-tubulin folding.” J. Biol. Chem. 289, 4490-4502 (Selected as paper of the week)
Lai, C. W. J., Kolesnikov, A.V., Frederick, J.M., Blake, D.R., Li, J., Stewart, J., Chen, C.-K., Barrow, J.R., Baehr, W., Kefalov, V.J. and Willardson, B.M. (2013) “Phosducin-like protein 1 is essential for G protein assembly and signaling in retinal rod photoreceptors.” J. Neurosci. 33, 7941-7951 (Selected in Faculty of 1000)
Smrcka, A. V., Kichik, N., Tarrago, T., Burroughs, M., Park, M., Stern, H., Itoga, N. K. Willardson, B. M. and Giralt, E. (2010) “NMR Analysis of G Protein βγ Subunit Complexes Reveals a Dynamic Gα-Gβγ Subunit Interface and Multiple Protein Recognition Modes” Proc. Natl. Acad. Sci. U. S. A. 107, 639-644.
Howlett, A. C., Gray, A. J., Hunter, J. M. and Willardson, B. M. (2009) “Role of Molecular Chaperones in G protein β5/Regulator of G protein Signaling Dimer Assembly and G protein βγ Dimer Specificity” J. Biol. Chem. 284, 16386-16399.
Lukov, G. L., Baker, C. M., Ludtke, P. J., Hu, T., Carter, M. D., Hackett, R. A., Thulin, C. D. and Willardson, B. M. (2006) “Mechanism of Assembly of G Protein βγ subunits by Protein Kinase CK2-phosphorylated Phosducin-like Protein and the Cytosolic Chaperonin Complex” J. Biol. Chem. 281, 22261-22274.
Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E. and Willardson, B. M. (2005) “Phosducin-like protein acts as a molecular chaperone for G protein βγ dimer assembly” EMBO J. 24, 1965-1975.
Martin-Benito, J., Bertrand, S., Hu, T., Ludtke, P., McLaughlin, J. N., Willardson, B. M., Carrascosa, J. L. and Valpuesta, J. M. (2004) “Structure of the complex between phosduin-like protein and the cytosolic chaperonin complex” Proc. Natl. Acad. Sci. 101, 17410-17415.
McLaughlin, J. N., Thulin, C. D., Hart, S. J., Resing, K. A., Ahn, N. G. and Willardson, B. M. (2002) “Regulatory Interaction of Phosducin-like Protein with the Cytosolic Chaperonin Complex” Proc. Natl. Acad. Sci. U.S.A. 99, 7962-7967.