Novel Activity of RGS14 on Go and Gi Nucleotide Binding and Hydr
详细信息 查看全文
- 作者：John R. Hepler ; Wendy Cladman ; Suneela Ramineni ; Susanne Hollinger ; and Peter Chidiac
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：April 12, 2005
- 年：2005
- 卷：44
- 期：14
- 页码：5495 - 5502
- 全文大小：596K
- 年卷期：v.44,no.14(April 12, 2005)
- ISSN：1520-4995
文摘
The bifunctional protein RGS14 is both a GTPase activating protein (GAP) for Gi
and Goand a guanine nucleotide dissociation inhibitor (GDI) for Gi. This GDI activity is isolated to a regionof 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 Gito modulate nucleotide binding and hydrolysis by mechanisms distinct from its defined GDI activity. Inbrain pull-down assays, full-length RGS14 and R14-RBD/GL (but not the isolated RGS domain of RGS14)bind Go-GDP, Gi-GDP, and also G
. When reconstituted with M2 muscarinic receptors (M2) pluseither Gi or Go, RGS4 (which has no RBD/GL domain) and full-length RGS14 each markedly stimulatesthe 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 affinityof RGS4 for Gi and Go, suggesting a cooperative interaction between the RBD/GL domain, RGS4,and G. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assayswith purified Gi or Go, 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 domaincan bind inactive forms of Go and Gi to confer previously unappreciated activities that influence Gnucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDIactivity.
and Goand a guanine nucleotide dissociation inhibitor (GDI) for Gi. This GDI activity is isolated to a regionof 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 Gito modulate nucleotide binding and hydrolysis by mechanisms distinct from its defined GDI activity. Inbrain pull-down assays, full-length RGS14 and R14-RBD/GL (but not the isolated RGS domain of RGS14)bind Go-GDP, Gi-GDP, and also G. When reconstituted with M2 muscarinic receptors (M2) pluseither Gi or Go, RGS4 (which has no RBD/GL domain) and full-length RGS14 each markedly stimulatesthe 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 affinityof RGS4 for Gi and Go, suggesting a cooperative interaction between the RBD/GL domain, RGS4,and G. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assayswith purified Gi or Go, 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 domaincan bind inactive forms of Go and Gi to confer previously unappreciated activities that influence Gnucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDIactivity.