Des Renford, Professor of Medicine,
Executive Director, Molecular Cardiology Program
Victor Chang Cardiac Research Institute

http://www.victorchang.org.au

Molecular cell biology of the heart in health and disease

Understanding the molecular signalling pathways that operate within the heart will lead to better treatments for people with heart disease. We are focused on understanding the structure and function of G-protein-coupled receptors, which are fundamental to the signalling pathways operating in the heart. These receptors are already the targets of over 50% of all known drugs and are present on the surface of cells in the heart and in every other major tissue and organ of the body. They play an important role in regulating the body’s response to a variety of different stresses. We are particularly interested in the adrenergic receptor, which is receptive to activation by hormones, such as adrenaline, that are released during times of stress, and a related novel G-protein discovered by this laboratory, termed Gh (for high-molecular-weight G-protein). Both play an extremely important role in the development of abnormal thickening of heart muscle (cardiac hypertrophy) and in the development of heart-rhythm disturbances (cardiac arrhythmias).

Program 1: Structure and function of adrenergic receptors

Project Leader :  Prof Bob Graham (b.graham@victorchang.unsw.edu.au)

Within the adrenergic receptor program we are examining the structure and function of the adrenergic receptors (AR) at both the molecular and whole animal levels.  We have two rodent models of cardiac specific a_1A-AR over expression which we are utilising to examine the role of the a_1A-AR in cardiac physiology and pathophysiology.  At the molecular level we are focused on delineating the critical conformational changes with in adrenergic receptors associated with specific G-protein activation pathways and non-G-protein signalling such as phosphorylation and internalisation. In our endeavours to understand the mechanisms of ligand-induced activation and signal transduction of the adrenergic receptors we are using molecular biology, pharmacology, cell biology, biochemistry and molecular modelling techniques. In particular we are using receptor mutagenesis, cell signalling assays, radioligand binding, western blotting, protein phosphorylation and confocal microscopy.  Using these techniques has allowed us to contribute to the development a model of biogenic amine GPCR-activation, which involves movements of helix VI away from helix III towards helix V. Based on studies in which TMVI residues were substituted with cysteine to act as sensors, we have recently shown that in the b₂-AR and a_1B –AR, this movement likely involves segments of TMVI, both above and below a highly conserved proline residue (Pro³⁰⁹).  Our molecular modelling predicted that Pro³⁰⁹ forms part of a kink in the middle of the helix and thus may play an important role in helix movement.  We are currently investigating the role of this proline in receptor activation; in addition, we have several unique mutant receptors that are allowing us to study the conformational changes associated with specific signalling pathways.

Selected References (Available on request)

• Chen, S., F. Lin, et al  (2002)  “Phe(303) in TMVI of the alpha (1B)-adrenergic receptor is a key residue coupling TM helical movements to G-protein activation.”  Biochemistry 41(2):588-96

• Chen, S., F. Lin, et al (2000)  “Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation.”  Embo J 19(16): 4256-71

• Chen, S., F. Lin, et al (2002).  “Mutation of a single TMVI residue, Phe(282), in the beta(2)-adrenergic receptor results in structurally distinct activated receptor conformations.”  Biochemistry 41(19): 6045-53

• Du, X.J., L. Fang, et al (2004)  “Genetic enhancement of ventricular contractility protects against pressure-overload-induced cardiac dysfunction.”  J Mol Cell Cardiol 37(5):979-87

• Lin, F., W.A. Owens, et al. (2001)  “Targeted alpha(1A)-adrenergic receptor overexpression induces enhanced cardiac contractility but not hypertrophy.”  Circ Res 89(4): 343-50.

Program 2: Structure and function of Gh

Project Leader: Dr Siiri Iismaa (s.iismaa@victorchang.unsw.edu.au )

Gh functions not only as a G-protein, but also as a post-translational modification enzyme that catalyses protein cross-linking (transglutaminase activity). Gh is thus also widely known as Transglutaminase type 2 (TG2), reflecting its dual roles. To elucidate the function and regulation of this protein we are utilising a wide range of techniques, including genetics, cell biology, molecular biology, protein biochemistry, biophysics and molecular modelling. This program has two major avenues of research. Firstly, we have generated a TG2 knock-out (KO) mouse and have shown these mice have, impaired clearance of apoptotic cells, that fibroblast adhesion is impaired and that TG2 is involved in bone development. We have recently established an exciting new role for TG2 in facilitating wound healing. At the cellular level, fibroblasts, which are integrally involved in wound healing, are being analysed for signalling pathways that are altered in the TG2 KO relative to wild-type. At the whole animal level, the effects of TG2 on the rate of wound healing are being analysed in a skin punch biopsy model. Secondly, we are investigating the catalytic and regulatory mechanisms of TG2. To this end, we have identified residues that stabilise the transition-state of the transglutaminase catalytic reaction and residues involved in the binding of GTP, which allosterically inhibits transglutaminase activity. We are investigating the effect of various mutations of TG2 on its ability to switch from its role as a G-protein to a transglutaminase. All assays and protocols required to complete these projects are currently in use in the laboratory. Projects will be developed in consultation with Dr Siiri Iismaa according to the student’s interests.

Another project is available in collaboration with Dr Merridee Wouters, Biocomputing Program, Victor Chang Cardiac Research Institute.

Selected references (available on request)

• Iismaa, S.E., Wu, M.-J., Nanda, N., Church, W.B. and Graham, R.M. GTP-binding and signalling by G_h/transglutaminase II involves distinct residues and a unique binding pocket. J. Biol. Chem. 275: 18259-18265, 2000

• Nanda, N., Iismaa, S.E., Owens, W.A., Husain, A., Mackay, F. and Graham, R.M. Targeted inactivation of G_h/tissue transglutaminase II. J. Biol. Chem. 276: 20673-27678, 2001

• Iismaa, S.E., Holman, S., Wouters, M.A., Lorand, L., Graham, R.M., Husain, A. Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases. Proc Natl Acad Sci USA   100: 12636-12641, 2003

• Johnson K, Van Elten D, Nanda N, Graham RM, Terkeltaub R. Distinct transglutaminase II/TG2-independent and TG2-dependent pathways mediate articular chondrocyte hypertrophy. J. Biol. Chem. 2003 21:18824-18832

• Lorand L, Graham RM.  Transglutaminases: crosslinking enzymes with pleiotropic functions. Nature Reviews Mol. Cell Biol. 2003; 4:140-156.