Using siRNA to define functional interactions between melanopsin and multiple G Protein partners

Cellular and Molecular Life Sciences, Jun 2014

Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of ocular photoreceptors and mediate a range of non-image forming responses to light. Melanopsin is a G protein coupled receptor (GPCR) and existing data suggest that it employs a membrane bound signalling cascade involving Gnaq/11 type G proteins. However, to date the precise identity of the Gα subunits involved in melanopsin phototransduction remains poorly defined. Here we show that Gnaq, Gna11 and Gna14 are highly co-expressed in pRGCs of the mouse retina. Furthermore, using RNAi based gene silencing we show that melanopsin can signal via Gnaq, Gna11 or Gna14 in vitro, and demonstrate that multiple members of the Gnaq/11 subfamily, including Gna14 and at least Gnaq or Gna11, can participate in melanopsin phototransduction in vivo and contribute to the pupillary light responses of mice lacking rod and cone photoreceptors. This diversity of G protein interactions suggests additional complexity in the melanopsin phototransduction cascade and may provide a basis for generating the diversity of light responses observed from pRGC subtypes.

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Using siRNA to define functional interactions between melanopsin and multiple G Protein partners

Steven Hughes 0 2 3 Aarti Jagannath 0 2 3 Doron Hickey 0 2 3 Silvia Gatti 0 2 3 Matthew Wood 0 2 3 Stuart N. Peirson 0 2 3 Russell G. Foster 0 2 3 Mark W. Hankins 0 2 3 0 S. hughes a. Jagannath D. hickey S. N. Peirson ( 1 ) Nuffield Laboratory of Ophthalmology, University of Oxford , Oxford OX3 9DU, UK 2 M. Wood Department of anatomy, Physiology and Genetics, University of Oxford , Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, UK 3 a. Jagannath S. Gatti F. hoffman La Roche, ReD Research and Development , CNS Dta, Basel, Switzerland Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of ocular photoreceptors and mediate a range of non-image forming responses to light. Melanopsin is a G protein coupled receptor (GPCR) and existing data suggest that it employs a membrane bound signalling cascade involving Gnaq/11 type G proteins. however, to date the precise identity of the G subunits involved in melanopsin phototransduction remains poorly defined. here we show that Gnaq, Gna11 and Gna14 are highly co-expressed in pRGCs of the mouse retina. Furthermore, using RNai based gene silencing we show that melanopsin can signal via Gnaq, Gna11 or Gna14 in vitro, and demonstrate that multiple members of S. hughes and a. Jagannath contributed equally to this work. - Over the last decade it has become clear that photoreception within the mammalian retina is not restricted to the rod and cones of the outer retina [1, 2], but also extends to a small number of photosensitive retinal ganglion cells (pRGCs) expressing the blue light sensitive photopigment melanopsin (for review see [35]). these inner retinal photoreceptors provide information regarding environmental irradiance and mediate a range of non-image forming (NIF) responses to light including circadian entrainment, pupil constriction and the induction of sleep [1, 69], and may also contribute to visual pathways [1012]. Since their original description, multiple distinct subtypes of pRGC have been identified (M1-M5 type pRGCs), characterised by differing levels of melanopsin expression and the stratification of their dendrites in specific sublamina of the inner plexiform layer (IPL) (for review see [4, 13]). In addition to these anatomical differences, a range of functional differences are now known to exist between these cell types, including the sensitivity and kinetics of photoresponses [10, 1417], the nature of synaptic inputs received from the outer retina [18], and the isoforms of melanopsin that they express [19, 20]. these cell types also innervate different areas of the brain [16, 2124] and mediate different physiological responses to light [24] (for review see [13, 25]). the mechanisms of phototransduction in melanopsin expressing pRGCs are known to be markedly different from that of rod and cone photoreceptors, and more closely resemble an invertebrate-like phototransduction pathway (for review see [35, 26]). Stimulation of melanopsin leads to the activation of a membrane bound signalling cascade involving Gnaq/11 type G proteins, activation of phospholipase-C (PLC) and ultimately opening of downstream tRP type ion channels [2731]. Recent studies have confirmed the precise identity of the PLC isoform involved, PLC4, and also the identity of the downstream ion channels, tRPC6 and tRPC7 [32]. however, despite the strong evidence that melanopsin couples to Gnaq/11 type G proteins within pRGCs [29, 31] and following expression in cell line systems [3337], all previous studies have utilised pharmacological tools that fail to distinguish between the specific members of the Gnaq/11 family. as a result, the specific identity of the G protein sub-unit(s) involved in melanopsin phototransduction remains to be determined. the Gnaq/11 family contains four members, Gnaq, Gna11, Gna14 and Gna15 (Gna16 in humans) [38, 39]. Single cell PCR analysis indicates that mRNa for multiple members of this family can be detected within individual pRGCs (likely M1 type cells), with Gna14 the most commonly detected [31]. however, to date the expression and localisation of Gnaq/11 type G proteins within the retina and within specific subclasses of pRGCs has not been investigated. In this study we have used immunohistochemistry in combination with in vitro and in vivo siRNa based gene silencing techniques to determine the specific G protein G subunits with which melanopsin is capable of interacting. We conclude that melanopsin has multiple G protein partners available within pRGCs and is capable of signalling via Gnaq, Gna11 and Gna14 G proteins in vitro and in vivo. expression of Gnaq/11 type G proteins in the mouse retina PCR and gene microarray analysis both show that mRNa transcripts for Gnaq, Gna11 and Gna14, but not Gna15 are expressed in the adult mouse retina (Supplementary Fig. 1). Given this profile of expression, we sought to determine the expression and localisation of Gnaq, Gna11 and Gna14 protein in the mouse (...truncated)


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Steven Hughes, Aarti Jagannath, Doron Hickey, Silvia Gatti, Matthew Wood, Stuart N. Peirson, Russell G. Foster, Mark W. Hankins. Using siRNA to define functional interactions between melanopsin and multiple G Protein partners, Cellular and Molecular Life Sciences, 2014, pp. 165-179, Volume 72, Issue 1, DOI: 10.1007/s00018-014-1664-6