Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins.
To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.
Ben-Yair
Liora
L
Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, Institute of Biochemistry, The Hebrew University, Rehovot 76100, Israel.
Slaaby
Rita
R
Herman
Asael
A
Cohen
Yael
Y
Biener
Eva
E
Moran
Nava
N
Yoshimura
Akihiko
A
Whittaker
Jonathan
J
De Meyts
Pierre
P
Herman
Brian
B
Gertler
Arieh
A
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000032:analysis*; D018613:Microscopy, Confocal; D010928:Placental Lactogen / Q000378:metabolism; D011388:Prolactin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D012756:Sheep; D015398:Signal Transduction; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014162:Transfection
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C108944:SOCS1 protein, human; C106643:SOCS2 protein, human; C106644:SOCS3 protein, human; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D011388:Prolactin; D010928:Placental Lactogen