Novel activity of RGS14 on Goalpha and Gialpha nucleotide binding and hydrolysis distinct from its RGS domain and GDI...

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• RGS14 is a bifunctional protein with GTPase Activating Protein (GAP) and Guanine Nucleotide Dissociation Inhibitor (GDI) activities.
• Its GDI activity resides in a region distinct from the RGS domain, known as the RGS domain that contains an additional G protein-binding domain (RBD/GL).

Purpose of the Study:

• To investigate the functions of RGS14 independent of its RGS domain.
• To characterize the role of the RGS domain-missing R14-RBD/GL in modulating G-protein activity.

Main Methods:

• Brain pull-down assays to assess binding of RGS14 variants to G-proteins.
• Reconstitution assays with M2 muscarinic receptors, G-proteins (Gi or Go), and RGS proteins to measure GTPase activity.
• Membrane-based and solution-based GAP assays to evaluate RGS4 and R14-RBD/GL interactions.

Main Results:

• RGS14 lacking its RGS domain (R14-RBD/GL) directly binds Goalpha-GDP, Gialpha-GDP, and Gbetagamma.
• R14-RBD/GL exhibits minimal direct GAP activity but potentiates RGS4's GAP activity by increasing its affinity for Gialpha and Goalpha.
• This potentiation requires membranes and/or receptors, as it's not observed in solution assays.

Conclusions:

• Regions of RGS14 outside the RGS domain bind inactive G-proteins (Gi and Go).
• These regions confer novel activities influencing G-protein nucleotide binding and hydrolysis through mechanisms distinct from the RGS domain and known GDI activity.
• RGS14 possesses previously unappreciated regulatory functions mediated by its non-RGS domains.