Dr Aylin Hanyaloglu
Regulation of G protein-coupled receptor (GPCR) signalling
Precise control of cellular communication and signalling are crucial for normal development and fertility in humans. The reproductive system co-ordinates signalling pathways in many different cell types to control oogenesis and spermatogenesis, ovulation, implantation, menstruation and parturition. Specific hormones initiate a cascade of cellular events that play a central role in these physiological processes and many of these function by activating specific G protein-coupled receptors (GPCRs) in target tissues. GPCRs represent the largest family of cell surface receptors and are activated by a variety of stimuli including light, ions, odorants, neurotransmitters and hormones. Their diversity means they play key physiological roles and their dysfunction underlies many pathological conditions, thus they are the focus of many drug design programs due to their fundamental biological and clinical importance.
By choreographing diverse receptor fates, the endocytic pathway plays a critical role in dictating receptor-signalling responses. A simple but dramatic example is the sorting of internalised GPCRs between lysosomal and recycling pathways, divergent traffic routes which produce essentially opposite effects on cell signalling (Figure 1).
Figure 1. Sorting of endocytosed GPCRs between divergent downstream pathways produces distinct cellular signalling patterns. Sorting of internalised receptors to lysosomes promotes receptor degradation and terminates receptor signalling, thus producing a prolonged attenuation of cellular signalling. In contrast, sorting of internalised receptors into a rapid recycling pathway allows cells to respond repeatedly to extracellular ligand. Under conditions of chronic agonist exposure can result in re-routing of ‘recycling’ receptors to the lysosomal pathway that contributes to receptor down-regulation, a trafficking fate thought to underlie the phenomenon of drug tolerance or tachyphylaxis (from Hanyaloglu and von Zastrow, 2008; Ann Rev Pharmacol Toxicol).
Mechanisms regulating the cellular fate of GPCRs are not only critical for dictating the hormonal responsiveness at the tissue level, but could also orchestrate and compartmentalise distinct signalling pathways responsible for a vast array of physiological responses. However, the identity of GPCR-associated sorting complexes and how this regulates cellular signalling in vivo remains elusive. We have recently identified a molecular pathway, which demonstrates that GPCR trafficking fate is critical in shaping the signalling response, and that adaptations in receptor trafficking may correspond to a specific physiological outcome (Figure 2). As disrupting the regulation of receptor signalling in humans underlies reproductive and metabolic disorders, such as polycystic ovarian syndrome, infertility, pre-term births and a number of endocrine cancers, a crucial goal is to understand the consequences of these molecular processes on both normal physiological function and in disease.
Figure 2. A multi-step model for sequence-directed recycling of the β2-adrenergic receptor, suggesting a hypothesis for how distinct GPCR trafficking fates could be specified. (a) Sequence-directed recycling of the β2-adrenergic receptor requires the EXEXXLL sequence, Hrs, and the PDZ-binding sequence. Hrs (blue) is proposed in the model to act as an endosome-associated scaffold, indirectly linked (by as-yet-unknown protein interactions) to the endocytosed receptor. (b) Disruption of the EXEXXLL sequence prevents receptors from engaging the endosome-associated scaffold and allows default recycling of receptors, independent of both Hrs and the PDZ-binding sequence. (c) Depletion of Hrs inhibits recycling of wild-type (but not EXEXXLL-mutant) β2-adrenergic receptors, resulting in receptor retention in the early endosome membrane. (d) Disruption of the PDZ-binding sequence specifies another endocytic itinerary, inhibited recycling of receptors with re-routing of internalized receptors to lysosomes. (from Hanyaloglu and von Zastrow, 2008; Ann Rev Pharmacol Toxicol).
The long-term goal of the group is to understand how hormone receptors regulate signal transduction of reproductive pathways, by studying the molecular basis of signal regulation from GPCRs. More specifically, we wish to understand how signal rapidity and specificity is achieved in the cell, by identifying the molecular networks associated with these receptors and how these regulate hormonal signalling in vivo.
Our program of work focuses on understanding how a cellular system exhibiting highly flexible regulation of trafficking fates for GPCRs confers distinct physiological responses in the reproductive pathway. Our previous work has studied the trafficking of the β2-adrenergic receptor, a prototypic GPCR that amongst other functions has crucial roles in relaxing the smooth muscle of the lung and the uterus, thus agonists to this receptor are clinically exploited in management of asthma and pre-term labour. The β2-adrenergic receptor undergoes rapid recycling following agonist-induced internalisation, a trafficking fate that is critical for signal recovery (Figure 1 and movie). GPCR recycling is an active process, involving multiple protein interactions.
Live imaging of GPCR trafficking following agonist activation using TIRF (total internal reflection fluorescence) microscopy. HEK-293 cells stably expressing FLAG-tagged β2-adrenergic receptors were treated with its agonist isoproterenol. The first event is receptor clustering in to clathrin-coated pits and internalisation into endosomes (larger round structures) some of which are highly motile. From some endosomes receptors entering budding recycling tubules are evident (from Hanyaloglu et.al, 2008, Mol Biol Cell).
We are also interested in expanding our knowledge of trafficking to other hormonal GPCR systems such as the gonadotrophin receptors. The group has expertise in, and access to, a wide repertoire of modern methodologies to study GPCR function, including genetic engineering, recombinant protein expression, cell culture, flow cytomtery, intracellular signalling, confocal imaging, FRET and BRET and mass spectrometry. Using a combination of these molecular, biochemical and physiological approaches our current work aims to:
- Identify the molecular machinery dictating the fate of GPCRs
- Analyse the consequences of these trafficking decisions on receptor signalling in vivo.
- To understand how changes in receptor sorting directly regulate hormonal sensitivity of target tissues.
Hanyaloglu AC, N'diaye EN, Kajihara KK, Puthenveedu MA, Wu P, von Zastrow M, Brown EJ (2008) The Ubiquitin-like Protein PLIC-2 Is a Negative Regulator of G Protein-coupled Receptor Endocytosis. Mol Biol Cell. 19(3):1252-1260
Hanyaloglu AC and von Zastrow M (2008) Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Ann Rev Pharmacol Toxicol. 10;48:537-568
Hanyaloglu AC and von Zastrow M (2007) A novel sorting sequence in the b2-adrenergic receptor switches recycling from default to the Hrs-dependent mechanism. J Biol Chem. 282(5):3095-104
Hanyaloglu AC, Mcullagh E, von Zastrow M (2005) Essential role of Hrs in a recycling mechanism mediating functional resensitization of cell signaling. EMBO J 24(13): 2265-83
Ward BK, Magno AL, Davis EA, Hanyaloglu AC, Stuckey BG, Burrows M, Eidne KA, Charles AK, Ratajczak T (2004) Functional deletion of the calcium-sensing receptor in a case of neonatal severe hyperparathyroidism. J Clin Endocrinol Metab 89(8):3721-30
Miles LE, Hanyaloglu AC, Dromey JR, Pfleger KD, Eidne KA (2004) Gonadotropin-releasing hormone receptor-mediated growth suppression of immortalized LbetaT2 gonadatrope and stable HEK 293 cell lines. Endocrinology 145(1):194-204
Hanyaloglu AC, Seeber R, Kohout TA, Lefkowitz RJ and Eidne KA (2002) Homo and hetero-oligomerization of thyrotropin-releasing hormone receptor (TRH) receptor subtypes-differential regulation of beta-arrestins 1 and 2. J Biol Chem 277(52), 50422-30
Eidne KA, Kroeger KM and Hanyaloglu AC (2002) Applications of novel resonance energy transfer techniques to study dynamic receptor interactions in living cells. Trends Endocrinol Metab 13(10):415-21. Review.
Hanyaloglu AC, Vrecl M, Kroeger KM, Miles LEC, Qian H, Thomas WG and Eidne KA (2001) Casein Kinase II sites in the intracellular C-terminal domain of the TRH receptor and chimeric GnRH receptors contribute to b-arrestin-dependent internalization. J Biol Chem 276(21):18066-18074
Kroeger KM, Hanyaloglu AC, Seeber R, Miles LEC and Eidne KA (2001) Constitutive and agonist-dependent oligomerization of the thyrotropin-releasing hormone receptor: detection in living cells using bioluminescence resonance energy transfer (BRET). J Biol Chem 276(16):12736-43.