TRANSMEMBRANE IV OF SECRETIN RECEPTOR AS A MOLECULAR DETERMINANT IN SECRETIN AND ANGIOTENSIN II TYPE 1A RECEPTOR DIMERIZATION

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 389-408-Signaling Originating from Membrane Receptors
Basic/Translational
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-401
Stephanie Yuen Lam Ng*1, Leo Tsz On Lee1, Kaleeckal G Harikumar2, Laurence J Miller2 and Billy Kwok Chong Chow1
1The University of Hong Kong, Hong Kong SAR, China, 2Mayo Clinic, Scottsdale, AZ
Osmoregulation is critical to life and is tightly regulated by integrated physiological and behavioral responses to maintain the osmolarity of fluid bathing cells. In particular, this involves recovery from dehydration both at the intracellular and extracellular levels. To achieve appropriate body fluid balance, three major hormones namely secretin (SCT), angiotensin II (ANGII) and vasopressin (VP) are responsible. Of note, SCT and ANGII share overlapping physiological roles including similar locality within the brain, dipsogenic actions and activation of VP expression and/or release in mice. However, it remains unclear how their receptor pathways may cross-interact to aid osmoregulation. In recent years, G protein-coupled receptor (GPCR) dimerization has been implicated to play roles in regulating processes such as expression, pharmacological diversity, signal transduction and internalization. Though not as extensively studied, class B GPCRs are also gaining merit in their dimerizing abilities, within which the wealth of available information is focused on homo- and hetero- dimerization of the secretin receptor (SCTR). On the basis of this information, our project aims to explore the molecular association between SCTR and ANGII receptors via in vitro experiments. In our study, bioluminescence resonance energy transfer (BRET) assays revealed mouse SCTR (mSCTR) and mouse ANGII type 1a receptor (mAT1a) to heterodimerize. This heterodimerization event was found by BRET competition to be contributed predominantly by mSCTR transmembrane (TM) domain regions 2, 4 and 7. Specifically, since TM4 has been identified in a previous study as a key homodimerization domain in mSCTR, we have examined the possible contribution of residues within TM4 via mutagenesis of the intact mSCTR. BRET assay revealed Val289 and Thr290 as potential molecular determinants in disrupting heterodimerization. Functionally, cAMP assay revealed heterodimerization to affect signaling negatively, reducing maximal response values by 25.9 ± 3.1 % compared to CHO-K1 cells transfected with only mSCTR. Moreover, with co-application of mouse SCT and ANGII peptides restoring cAMP maximal response to 92.6 ± 5.3 % only when mAT1a was co-expressed with mSCTR and not alone (11.6 ± 0.9 %), this suggests heterodimerization to be necessary for mAT1a to signal via the cAMP pathway. Taken together, our data on mSCTR and mAT1a heterodimerization may provide some molecular clues to the overlapping roles of SCT and ANGII in water homeostasis.

(1) Chu, J.Y., Lee, L.T., Lai, C.H., Vaudry, H., Chan, Y.S., Yung, W.H., and Chow, B.K. (2009). Secretin as a neurohypophysial factor regulating body water homeostasis. Proc Natl Acad Sci U S A 106, 15961-15966. (2) Lee, V.H., Lee, L.T., Chu, J.Y., Lam, I.P., Siu, F.K., Vaudry, H., and Chow, B.K. (2010). An indispensable role of secretin in mediating the osmoregulatory functions of angiotensin II. FASEB J 24, 5024-5032. (3) Harikumar, K.G., Pinon, D.I., and Miller, L.J. (2007). Transmembrane segment IV contributes a functionally important interface for oligomerization of the Class II G protein-coupled secretin receptor. J Biol Chem 282, 30363-30372.

Nothing to Disclose: SYLN, LTOL, KGH, LJM, BKCC

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: (1) Hong Kong Government RGC grants GRF764510: An Indispensable Role of Secretin in Mediating Effects of Angiotensin-II in the Brain; CRFHKU6/CRF/11G: Strategic research of hormones and their receptors in the water homeostatic axis: from molecular mechanisms to anti-hypertensive drug design awarded to Billy K.C. Chow (2) Hong Kong Government RGC grant GRF770212: Secretin and secretin receptor as key regulators of cardiovascular functions awarded to Leo T.O. Lee