Selective interactions between Gi alpha1 and Gi alpha3 and the GoLoco/GPR domain of RGS14 influence its dynamic subcellular localization.
RGS14 is a multifunctional protein that contains an RGS domain, which binds active Gi/o alpha-GTP, a GoLoco/GPR domain, which binds inactive Gi alpha-GDP, and a tandem Rap1/2 binding domain (RBD). Studies were initiated to determine the roles of these domains and their interactions with Gi alpha on RGS14 subcellular localization. We report that RGS14 dynamic subcellular localization in HeLa cells depends on distinct domains and selective interactions with preferred Gi alpha isoforms. RGS14 shuttles rapidly between the nucleus and cytoplasm, and associates with centrosomes during interphase and mitosis. RGS14 localization to the nucleus depends on the RGS and RBD domains, its translocation out of the nucleus depends on the GoLoco/GPR domain, and its localization to centrosomes depends on the RBD domain. Gi alpha subunits (Gi alpha1, 2 and 3) localize predominantly at the plasma membrane. RGS14 binds directly to inactive and active forms of Gi alpha1 and Gi alpha3, but not Gi alpha2, both as a purified protein and when recovered from cells. RGS14 localizes predominantly at the plasma membrane in cells with inactive Gi alpha1 and Gi alpha3, but not Gi alpha2, whereas less RGS14 associates with active Gi alpha1/3 at the plasma membrane. RGS14 binding to inactive, but not active Gi alpha1/3 also prevents association with centrosomes or nuclear localization. Removal or functional inactivation of the GoLoco/GPR domain causes RGS14 to accumulate at centrosomes and in the nucleus, but renders it insensitive to recruitment to the plasma membrane by Gi alpha1/3. These findings highlight the importance of the GoLoco/GPR domain and its interactions with Gi alpha1/3 in determining RGS14 subcellular localization and linked functions.
Shu
Feng-jue
FJ
Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322-3090, United States.
Ramineni
Suneela
S
Amyot
Whitney
W
Hepler
John R
JR
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008938:Mitosis; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020710:RGS Proteins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D020710:RGS Proteins; D011994:Recombinant Proteins; C106374:Rgs14 protein, rat; D019206:GTP-Binding Protein alpha Subunits, Gi-Go