Urinary incontinence (UI) is an increasing medical problem in ageing populations. Affecting more than 12 million afflicted people in the US alone, UI is a frequent cause of confinement and lifestyle modification [1]. UI is defined as the involuntary loss of urine, and may result from a number of causes including the improper control of detrusor activity or compromised urethral function. UI can also occur as a complication of other diseases such as Parkinson disease, multiple sclerosis, and bladder infections, indicating that there are both muscular and neuronal components of the disease.
Current treatments for UI rely on antagonism of G-protein coupled receptors (GPCRs) of the muscarinic acetylcholine receptor class [2]. The signal transduction pathways downstream of muscarinic GPCRs are responsible for bladder muscle cell contractility, and antagonists of these receptors allow for greater bladder filling. While muscarinic GPCR antagonists are generally safe, they have unwanted side effects due to the broad tissue expression of their targets [3–5]. GPCRs are the most successful class of targets for disease states including hypertension, diabetes, obesity, depression, osteoporosis, and inflammation. In fact, more than half of currently marketed drugs for the condition act as modulators of this protein class [6,7]. Methods to modulate other signaling nodes downstream of GPCRs may hold potential for safer and more efficacious therapies.
Heterotrimeric G-proteins are the proximal signaling partners downstream of GPCRs. Binding of acetylcholine to the muscarinic GPCRs results in the exchange of GDP for GTP on the G-protein α subunit (G-αq). This activation event allows dissociation of G-αq from the G-β/γ heterodimer. The dissociated G-protein subunits then mediate separate cellular responses through their interactions with enzymes, channels, kinase cascades, and intracellular second messengers [8–10]. In smooth muscle cells, activation of G-αq results in protein kinase C (PKC)-dependent calcium mobilization and subsequent muscle contraction. Following GTP hydrolysis on G-α, the heterotrimeric G-protein complex reforms and signaling is terminated. G-protein function is under the strict control of factors such as the regulators of G-protein signaling (RGS) proteins. RGS proteins were first identified as potent negative regulators of GPCR signaling in yeast [11], and are now known to act as GTPase activating proteins (GAPs) in all eukaryotic systems. RGS proteins bind directly to the G-α subunit and enhance the rate of GTP hydrolysis, thereby shortening the lifetime of the dissociated, active G-protein species and curtailing GPCR signaling [12]. At least 24 mammalian proteins contain a common “RGS core domain” [13]. Interestingly, many RGS proteins have been shown to have spatially restricted expression patterns, suggesting that they may allow tissue-specific control of ubiquitous G-proteins [14,15].
In this paper we describe mechanism-of-action studies with small molecules that were originally identified by their activity in an ex vivo bladder contraction assay. These effects are mediated by a heretofore unknown molecular mechanism [16]. Notably, in vivo studies using a selected example (BMS-195270) revealed that this small molecule displayed marked tissue specificity, inhibiting bladder contractility at doses that did not significantly affect blood pressure or heart rate [16] (unpublished data). To define the pathway of action of these small molecules, we have used genetic screens in Caenorhabditis elegans coupled with biochemical assays in mammalian systems. We demonstrate that these small molecules likely act at the intersection of RGS and G-αq proteins, resulting in the downregulation of GPCR signaling, reduced calcium fluxes, and reduced muscle contraction. In addition, we have uncovered novel mutations in RGS and G-αq proteins, including the first hypo-adaptation allele of a G-αq protein. Identification of a novel set of compounds that function to limit signaling downstream of GPCRs, as well as a hypo-adapation allele of G-αq, has implications for the many diseases currently treated via direct modulation of these receptors.
Answers to all your Biology Questions
