DETERMINATION OF ANGIOTENSIN II TYPE 2 RECEPTOR PROTEIN INTERACTIONS USING THE YEAST-TWO HYBRID SYSTEM

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Emily Janeen Westover and Dr. Terry S. Elton, Chemistry and Biochemistry

The renin-angiotensin system plays a central role in water and salt homeostasis, electrolyte balance, and vascular tone maintenance. 1 Each component of this system is a potential effector in the etiology of hypertension. Angiotensin II (Ang II), a component of the reninangiotensin system, interacts with two distinct receptor types, the Angiotensin Type 1 Receptor (AT1R) and the Angiotensin Type 2 Receptor (AT2R). The receptor types 2 probably mediate opposite intracellular effects.3 However, all currently known effects of Ang II involve the AT1R. Because of its novelty and importance, we are investigating the 4 role of the AT2R by determining proteins that interact with this receptor.

The yeast two-hybrid system, developed by Dr. Stanley Fields, provides a method for studying protein-protein interactions.5 This system uses specific eukaryotic transcription activators in a special yeast strain transformed with two plasmids. Eukaroytic transcription activators require that two distinct domains—the DNA Binding Domain (BD) and the Activating Domain (AD)-come together to initiate transcription. Their activity can be assayed by the production of a reporter gene product. (Fig. 1) Thus, the amount of reporter formed indicates the strength of the interaction between the fusion protein and the library gene.

For our studies we used the L40 yeast strain. We ligated the AT2R cDNA clone into the pBTM116 vector in frame with the Lex A-DNA Binding Domain. This fusion protein allows for interaction with the Lex A operator. We also used a fetal mouse CDNA library, constructed in the pVPI6 plasmid. This library contains many different plasmids in which CDNA is ligated in frame with the Lex A-Activating Domain. This fusion protein brings the Activating Domain in proximity to the Binding Domain if the library protein interacts with the AT2R.

Once the yeast was transformed with both plasmids, we analyzed which proteins from the fetal mouse library interact with the AT2R. When the Lex A Binding Domain-AT2R/Lex A Activating Domain-Library Protein complex binds to the Lex A operator, production of the lac z reporter increases. Any protein domain encoded by the mouse CDNA library that binds to the AT2R will increase lac z production. (Fig. 1)

A -galactosidase assay was used to identify strong protein interactions with the AT2R. cDNA clones which produced dark blue colonies on X-gal plates were studied further. Plasmid DNA from the dark blue colonies was isolated, PCR amplified, subcloned, and sequenced. The sequences were analyzed for sequence homology with known proteins through Genebank. Preliminary results indicate that the AT2R interacts with a nucleosome assembly protein and a ribosomal protein. Several clones are being sequenced further, pending analysis through Genebank. The CDNA clones with homology to known proteins are currently being further characterized. Independent methods will be used to substantiate all putative interactions.

References

  1. Hall, J. E. and A.C. Guyton. (1990). “In Hypertension: Pathophysiology, Diagnosis, and Management” 1105-1129.
  2. Chang, R. S. L., Lotti, V. J., Chen, T. B. and Faust, K. A. Biochem. Biophys. Res. Comm. 171,813-817.
  3. Elton, T. S., unpublished observations.
  4. Murphy, T. J. et al. (1991). Nature 351, 233-236.
  5. Fields, S. and Song. (1989). Nature 340, 245-246.
  6. The help of Mickey Martin is gratefully acknowledged.