Gb5-RGS Complexes

We entered the RGS field unexpectedly while pursuing the identification of in vivo binding partners of Gb 5, a neurospecific G protein b subunit. Upon purification of a native Gb 5 complex, we found that instead of interacting with specific Ga and Gg subunits, it is bound to RGS7. This discovery showed for the first time that a Gb subunit can exist without a Gg subunit and demonstrated that an RGS can interact with a protein other than Ga. Since these mechanisms were unknown, we concentrated our efforts on further studies of Gb 5-RGS complexes. Our analysis of brain Gb 5-RGS7 and photoreceptor-specific Gb 5L-RGS9 has demonstrated the following:

1. Gb 5-RGS dimers are present in vivo and can be reconstituted in vitro.
2. The Gb 5-RGS interaction is specific for Gb 5 and is determined by the Gg-like (GGL) domain found in a distinct RGS subclass represented in mammals by RGS 6, 7, 9 and 11.
3. In native tissues, Gb 5 and RGS are only found in the complex. In other words, Gb 5-Gg nor monomeric RGS of this type do not exist in vivo. Moreover, in vitro, Gb 5 preferentially binds RGS7 in the presence of excess Gg2.
4. Cells maintain the Gb 5:RGS stoichiometry via degradation of the unassociated proteins.
5. In vitro, the Gb 5-RGS7 complex cannot bind to Ga with high affinity, but in cell-based assays, the dimer can still inhibit G protein signaling by stimulating the GTPase activity of Ga .

Previous studies by many labs established fundamental role of "classical" Gbg complexes as very important adapter molecules that directly associate with receptors, Ga subunits, effectors, and a number of regulatory proteins. These interactions are uniquely important in signal transduction and crosstalk between various pathways. The structure of the Gb5-RGS dimers is significantly more complex than that of Gbg, indicating that the pattern of their interactions can be even more intricate. We anticipate that studies of Gb5-RGS function will develop into a new dynamic and multifaceted area of neuronal signaling. This research is challenging because we do not know the specific receptor-G protein-effector pathways that Gb 5-RGS belong to. Our first step in understanding the physiologic role of these dimers is to identify molecules that interact with Gb 5-RGS in vivo. For this purpose, we are testing potential interactions using a variety of biochemical and biophysical methods, cell-based assays, as well as genetic approaches.

Keren-Raifman T, Bera AK, Zveig D, Peleg S, Witherow DS, Slepak VZ, Dascal N. Expression levels of RGS7 and RGS4
proteins determine the mode of regulation of the G protein-activated K(+) channel and control regulation of RGS7 by G beta 5. FEBS Lett. 2001 Mar 9;492(1-2):20-8.

Witherow, D. S., Wang, Q., Levay, K, Cabrera. J. L., Chen, J., Willars, G. and Slepak, V. Z. "Complexes of the G protein subunit Gb5 with regulators of G protein signaling RGS7 and RGS9: characterization in native tissues and in transfected cells" J. Biol. Chem. (2000), 275, 24872-24880.

Levay, K. Cabrera, J. L. Satpaev, D. K. and Slepak, V.Z. "Gb5 prevents the RGS7-Gao interaction through binding to a distinct Gg-like domain found in RGS7 and other RGS proteins" Proc. Natl. Acad. Sci. (1999) 96, 2503-2507.

Cabrera, J. L. de Freitas, F., Satpaev D. K., and Slepak, V.Z. "Identification of the Gb5-RGS7 Protein Complex in the Retina." Biochim. Biophis. Res. Commun. (1998), 249, 898-902.

Interview with Gb5: A casual conversation with this unusual Gb subunit recorded by Scott Witherow and Vlad Slepak.