Part:BBa_K4875003

α2A-AR-CT

Profile: α2A-AR-CT

Profile Details

• Name: α2A-AR-CT
• Base Pairs: 72 bp
• Origin: Synthetic
• Properties: It is the carboxyl terminal of the 2A adrenergic receptor in the G protein coupled receptor (GPCR) family

Usage and Biology

The α2-adrenoceptor (α2-AR) family comprises α2A-, α2B- and α2C-AR subtypes [1-3], which are expressed in presynaptic membranes of adrenergic neurons, where they act as autoreceptors (receptors stimulated by the neurotransmitter released by the neuron where they are located. Stress activates BNST CRF neurons, and that α2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.[4]

Stress activates BNST CRF neurons, and that α2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.

Cultivation, Purification and SDS-PAGE

We used primers to amplify α2A-AR fragment from α2A-AR-GFP and inserted it into the vectors pGEX-4T-1 to obtain GST-α2A-AR-CT plasmid. At the end of the experiment, we obtained the target protein by purification, and we compared it with the sample from each process during purification by using protein gel electrophoresis. The electrophoresis result showed that our target protein now became pure, and we finally identified that we got the right protein (25 kDa).

Figure 1: Schematic diagram of GST-α-AR-CT protein purification. Image by Xu Xing.[5]

Figure 2: SDS-PAGE analysis of GST-α-AR-CT

Characterization/Measurement

Through the GO data, we can know that our target gene is enriched at the biological process level, with the highest number of genes enriched in translation, and our differential genes are most significantly enriched in this regulation of translation. Through the KEGG data, we can know that the number of genes enriched on Ribosome is the highest, and our differential genes are most significantly enriched in Thermogenesis.

Figure 3: KEGG Pathways for Significantly Expressed Proteins

Figure 4: GOBP for Significantly Expressed Proteins

Based on the IP-MS results in figure 3 and 4, we could further find our candidate proteins that have the potential ability to affect the α-AR-CT transportation to the membrane, and the detailed result could be found in this EXCEL.

IP-MS was completed by Professor Shen's research group at Nanjing University of Traditional Chinese Medicine, providing IP-MS testing service and data analysis service.

References

1. Latifpour J, Jones SB, Bylund DB. Characterization of [3H]yohimbine binding to putative alpha-2 adrenergic receptors in neonatal rat lung. J Pharmacol Exp Ther. (1982) 223:606–11.
2. Kobilka BK, Matsui H, Kobilka TS, Yang-Feng TL, Francke U, Caron MG, et al. Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor. Science (1987) 238:650–6. 10.1126
3. Knaus AE, Muthig V, Schickinger S, Moura E, Beetz N, Gilsbach R, et al. Alpha2-adrenoceptor subtypes–unexpected functions for receptors and ligands derived from gene-targeted mouse models. Neurochem Int. (2007) 51:277–81.
4. Fetterly TL, Basu A, Nabit BP, Awad E, Williford KM, Centanni SW, Matthews RT, Silberman Y, Winder DG. α2A-Adrenergic Receptor Activation Decreases Parabrachial Nucleus Excitatory Drive onto BNST CRF Neurons and Reduces Their Activity In Vivo. J Neurosci. 2019 Jan 16;39(3):472-484.
5. Xu X, Wu G. Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport. J Biol Chem. 2022 Jun;298(6):102021.

Sequence and Features




Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]