Regulators of G protein signalling proteins in the human myometrium

doi:10.1016/j.ejphar.2009.03.042

European Journal of Pharmacology

Volume 610, Issues 1–3, 21 May 2009, Pages 23-28

https://doi.org/10.1016/j.ejphar.2009.03.042 Get rights and content

Abstract

The contractile state of the human myometrium is controlled by extracellular signals that promote relaxation or contraction. Many of these signals function through G protein-coupled receptors at the cell surface, stimulating heterotrimeric G proteins and leading to changes in the activity of effector proteins responsible for bringing about the response. G proteins can interact with multiple receptors and many different effectors and are key players in the response. Regulators of G protein signalling (RGS) proteins are GTPase activating proteins for heterotrimeric G proteins and help terminate the signal. Little is known about the function of RGS proteins in human myometrium and we have therefore analysed transcript levels for RGS proteins at various stages of pregnancy (non-pregnant, preterm, term non-labouring, term labouring). RGS2 and RGS5 were the most abundantly expressed isolates in each of the patient groups. The levels of RGS4 and RGS16 (and to a lesser extent RGS2 and RGS14) increased in term labouring samples relative to the other groups. Yeast two-hybrid analysis and co-immunoprecipitation in myometrial cells revealed that both RGS2 and RGS5 interact directly with the cytoplasmic tail of the oxytocin receptor, suggesting they might help regulate signalling through this receptor.

Introduction

Initiation of labour involves a change from quiescence to regular, forceful uterine contractions. The change is mediated by hormonal, metabolic and intracellular signalling, but the regulatory mechanisms underlying this process are poorly understood. Information relating to these mechanisms is essential to providing a better understanding of disorders associated with human parturition, such as pre-term labour (Lopez-Bernal and TambyRaja, 2000). Many of the signalling pathways that regulate contraction and relaxation of myometrial cells involve G protein-coupled receptors (Europe-Finner et al., 1997, Plested and Lopez-Bernal, 2001). In the unstimulated state, the receptor is associated with a heterotrimeric complex of Gα, Gβ and Gγ subunits in which the Gα subunit is bound to GDP. Receptor activation stimulates exchange of GDP for GTP, releasing the Gα-GTP and Gβγ subunits to activate downstream effectors that bring about changes in cell behaviour. For example, the oxytocin receptor functions via G proteins that activate phospholipase C, thereby increasing inositol trisphosphate (IP3) production, leading to increases in intracellular calcium, triggering muscle contraction (Blanks and Thornton, 2003). The receptors for prostaglandins, vasopressin and corticotrophin-releasing factor (CRF) also act via G protein-coupled receptors and G proteins. As many G protein-coupled receptors can interact with more than one G protein, and many G proteins can activate more than one type of effector protein, the G proteins play a pivotal role in integrating the stimulatory and inhibitory signals that regulate contractility.

G protein signalling ceases when Gα-GTP hydrolysis returns the heterotrimer to its inactive state. The slow intrinsic rate of GTP hydrolysis by Gα proteins is regulated by interactions with a specific subfamily of GTPase-activating proteins (GAPs) known as Regulators of G protein Signalling or RGS proteins (Ross and Wilkie, 2000, Hollinger and Hepler, 2002, Xie and Palmer, 2007). Since their discovery in Caenorhabditis elegans (Koelle and Horvitz, 1996) and Saccharomyces cerevisiae (Dohlman et al., 1996), over 30 different RGS proteins have been identified in mammals, many with spatiotemporal-specific expression (Abramow-Newerly et al., 2006, Xie and Palmer, 2007).

There have been several studies into the ability of RGS proteins to regulate contraction in cardiomyocytes (Tamirisa et al., 1999, Mittmann et al., 2002, Snabaitis et al., 2005, Hao et al., 2006), vascular smooth muscle cells (Tang et al., 2003) and intestinal smooth muscle (Hu et al., 2008) but little is known about their role in myometrium. Microarray analyses have detected expression of several RGS transcripts in human myometrium and a recent study found that expression of RGS12 was upregulated at labour (O'Brien et al., 2008). Earlier studies from Soloff and colleagues showed that RGS2 mRNA levels increased in cultured human myometrial cells following stimulation with oxytocin (Park et al., 2002) while RGS2 transcription in rats increased dramatically during pregnancy before being down-regulated at term (Suarez et al., 2003). However, no investigation of the role of RGS2 was undertaken in either system.

To initiate a more complete understanding of RGS expression in human myometrium, we used semi-quantitative polymerase chain reaction (PCR) to analyse transcript levels for all of the major RGS proteins at various stages of pregnancy (non-pregnant, preterm, term non-labouring, term labouring). Transcripts for many of the RGS proteins were present at low level and did not vary throughout pregnancy, although the levels of RGS4 and RGS16 (and to a lesser extent RGS2 and RGS14) increased in the term labouring samples. RGS2 and RGS5 were the most abundantly expressed isolates in each of the patient groups and we sought to further investigate potential roles for these two RGS proteins in human myometrium. Yeast two-hybrid analysis and co-immunoprecipitation experiments in primary myometrial cells revealed that both RGS2 and RGS5 interact directly and specifically with the cytoplasmic tail of the oxytocin receptor. Our results suggest a potential role for RGS proteins in regulating signalling in the human myometrium.

Section snippets

Subject criteria and selection

All procedures were conducted within the guidelines of The Declaration of Helsinki and were subject to local ethical approval (REC-05/Q2802/107). Myometrial samples were collected with informed written consent from the following groups of women: (i) non-pregnant (aged 38–48 years) undergoing hysterectomy for dysmenorrhoea (ii) preterm pregnant women (aged 20–30 years) prior to the onset of labour (“preterm”) (iii) term pregnant women prior to labour (“term, non-labouring”) (iv) term pregnant

Expression of RGS1-RGS16 mRNA in human myometrium

Semi-quantitative RT-PCR was used to analyse RGS mRNA expression in different samples of human myometrium, and the results compared to the mRNA levels of calponin (Fig. 1). The mRNA level of calponin do not change with or throughout pregnancy (Brodt-Eppley and Myatt, 1999, Moore et al., 1999), providing an appropriate mechanism to control or indicate standardising the study. Nucleotide sequencing confirmed that the PCR products corresponded to the expected RGS targets (not shown). A comparison

Discussion

We report the first comprehensive analysis of RGS expression in human myometrium at different stages of pregnancy. RGS2 and RGS5 were highly expressed in all samples, while expression of RGS4, RGS14 and RGS16 increased in the term labouring samples. RGS2 levels were higher in the term labouring samples than in the preterm and term non-labouring samples, although it was also relatively high in the non-pregnant sample. Transcripts for RGS1, RGS9, RGS10, RGS12 and RGS13 were detected at low levels

Acknowledgments

This work was supported by Wellbeing (Project Grant 2198, ST and JD) and the University Hospitals of Coventry and Warwickshire NHS Trust (GL and ST). We thank the patients and staff at Women's Hospital, University Hospitals of Coventry and Warwickshire, Coventry for their help in collecting myometrial biopsies. In particular, we thank Jane Green who supervised sample collection.

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Molecular and Cellular PharmacologyRegulators of G protein signalling proteins in the human myometrium

Abstract

Introduction

Section snippets

Subject criteria and selection

Expression of RGS1-RGS16 mRNA in human myometrium

Discussion

Acknowledgments

Cell. Signal.

Cell

Pharmacol. Ther.

J. Biol. Chem.

J. Biol. Chem.

BJOG

Obstet. Gynecol.

J. Biol. Chem.

Signal

J. Biol. Chem.

J. Mol. Cell. Cardiol.

Cell. Signal.

Cell

Cell. Signal.

Baillieres Best Pract. Res., Clin. Obstet. Gynaecol.

Trends Pharmacol. Sci.

Am. J. Obstet. Gynecol.

Semin. Cell Dev. Biol.

Cell. Signal.

J. Biol. Chem.

Am. J. Obstet. Gynecol.

Semin. Cell Dev. Biol.

J. Biol. Chem.

Semin. Cell Dev. Biol.

Molecular and Cellular Pharmacology
Regulators of G protein signalling proteins in the human myometrium