Difference between revisions of "Part:BBa K2295000"

Revision as of 15:37, 1 November 2017

TDAG8 (T cell death associated gene 8)
TDAG8 (T cell death associated gene 8) is a proton sensing Gs coupled Receptor.

Overview

This part is a Gs coupled GPCR. It is pH sensitive, hence lowering pH should increase intracellular cAMP.

G protein coupled receptors in general

Figure 1:

TDAG8 signaling pathway

G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors or heptahelical receptors, are a large family of integral membrane proteins that respond to many different extracellular stimuli. The two principal signal transduction pathways involving GPCRs are the cAMP signal pathway as well as the phosphatidylinositol signal pathway.

Mechanism

Due to the fact that this BioBrick is mainly used for cAMP dependent transcription, only the cAMP cascade will be described, which is characteristical for Gs coupled GPCRs. Being activated by its extracellular ligand (Figure 1: H+ ions), a conformational change is induced in the receptor. This change is transmitted to an attached intracellular heterotrimeric G protein complex (Figure 1: Gs). Exchanging GDP for GTP due to the stimulation, the Gs alpha subunit is released from the complex (not shown in Figure 1 for didactic reasons). Binding to adenylyl cyclase (AC), Gs aplha subunit activates AC. This results in the catization of the conversion of ATP into cyclic adenosine monophosphate (cAMP). Although the increase of intracellular concentration of this secondary messenger has numoerous effectors, the main pathway of BBa_K2295001 continues with the activation of a cAMP dependent enzyme called protein kinase A (PKA) (Figure 1: Protein Kinase A). PKA phosphorylates a number of other proteins. It also translocates into the nucleus where it activates cAMP responsive binding elements (CREB). Being now phosphorylated, CREBs can bind to cAMP responsive elements such as the cre promoter (BBa_K2295001), activating downstream transcription.

Freiburg 2017's Characterization

The cAMP response element-containing promoter (pCRE), which is pH responsive, was characterized in vitro in Jurkat and HEK293T cell lines. For this purpose, the pH of the media had to be adjusted.

In order to characterize the CRE promoter, stably transduced HEK293T and Jurkat lines were created expressing eCFP under a minimal promoter with multiple CRE sites. Induction was performed with pH adjusted media. Constitutively expressed mCherry was used as transduction marker. For analysis in HEK293T, PEI transfection of the pH receptor TDAG8, which is not expressed in these cells, was performed. (Ausländer et al., 2014). To generate a high expression by activating the signaling cascade downstream of the receptor, the stable cell lines were induced with forskolin and IBMX. Forskolin activates the cAMP-producing enzyme adenylyl cyclase and IBMX inhibits cAMP-hydrolyzing phosphodiesterases (Bittinger et al., 2004). Fluorescence was measured by flow cytometry after 24 h of treatment (Fig. 2).

Figure 2: Flow cytometry of hypoxia response element promoter analysis. z) Jurkat cells stably transduced with 4xCRE-pTal:eCFP were incubated 24 h in pH adjusted RPMI 1640. z) The experiment was repeated inducing with Forskolin (10 µM) and IBMX (10 µM). a) and b) HEK293T cells stably transduced with 4xCRE-pTal:eCFP with and without transient TDAG8 were induced similarly to z) and z). Results show the represent the amount of eCFP positive cells a) and the b) mean fluorescence intensity. All data points are mean values of triplicates, error bars represent standard deviation. Significant differences in z) and z) were determined using ANOVA, for a) and b) significant differences were determined using one-tailed student’s t-test (Excel 2017); * p < 0.05, ** p < 0.01, *** p < 0.001, non-significant differences are not marked.

Sequencing Results

Sanger sequencing was done at GATC with BBa_G00100 and BBa_G00101. Files of the results can be found here: https://parts.igem.org/File:BBa_K2295000-psb1c3-tdag8-sequencing-files.zip

Sequence and Features