The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.
Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Ningfei
N
An
Joe B
JB
Blumer
Michael L
ML
Bernard
Stephen M
SM
Lanier
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D054731:PDZ Domains / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C532305:Frmpd1 protein, human; C532306:Frmpd1 protein, rat; C525615:GPSM1 protein, human; D044385:GTP-Binding Protein alpha Subunits; C498913:Gpsm1 protein, rat; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D018341:Receptors, Adrenergic, alpha-2; C028841:erythrocyte membrane band 4.1 protein; D005576:Colforsin