Novel activity of RGS14 on Goalpha and Gialpha nucleotide binding and hydrolysis distinct from its RGS domain and GDI activity.
The bifunctional protein RGS14 is both a GTPase activating protein (GAP) for Gialpha and Goalphaand a guanine nucleotide dissociation inhibitor (GDI) for Gialpha. This GDI activity is isolated to a region of the protein distinct from the RGS domain that contains an additional G protein-binding domain (RBD/GL). Here, we report that RGS14 missing its RGS domain (R14-RBD/GL) binds directly to Go and Gi to modulate nucleotide binding and hydrolysis by mechanisms distinct from its defined GDI activity. In brain pull-down assays, full-length RGS14 and R14-RBD/GL (but not the isolated RGS domain of RGS14) bind Goalpha-GDP, Gialpha-GDP, and also Gbetagamma. When reconstituted with M2 muscarinic receptors (M2) plus either Gi or Go, RGS4 (which has no RBD/GL domain) and full-length RGS14 each markedly stimulates the steady-state GTPase activities of both G proteins, whereas R14-RBD/GL has little or no effect. R14-RBD/GL potentiates RGS4 GAP activity in membrane-based assays by increasing the apparent affinity of RGS4 for Gialpha and Goalpha, suggesting a cooperative interaction between the RBD/GL domain, RGS4, and Galpha. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assays with purified Gialpha or Goalpha, suggesting that membranes and/or receptors are required for this activity. When these findings are taken together, they indicate that regions of RGS14 outside of the RGS domain can bind inactive forms of Go and Gi to confer previously unappreciated activities that influence Galphanucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDI activity.
Hepler
John R
JR
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322-3090, USA. jhepler@emory.edu
Cladman
Wendy
W
Ramineni
Suneela
S
Hollinger
Susanne
S
Chidiac
Peter
P
D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D020730:Guanine Nucleotide Dissociation Inhibitors; D009711:Nucleotides; D020710:RGS Proteins; C491400:RGS14 protein, human; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins