Using siRNA to define functional interactions between melanopsin and multiple G Protein partners
Steven Hughes
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Aarti Jagannath
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Doron Hickey
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Silvia Gatti
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Matthew Wood
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Stuart N. Peirson
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Russell G. Foster
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Mark W. Hankins
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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
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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.
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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)