1 MINI-REVIEW

2 Spatial programming of -coupled receptor activity:

3 decoding signalling in health and disease

4 Camilla West and Aylin C. Hanyaloglu*

5 Institute of Reproductive Biology and Development, Department of Surgery and Cancer, Imperial

6 College London, UK

7

8 *Corresponding author and to whom reprint requests should be addressed: Dr. Aylin

9 Hanyaloglu, Institute of Reproductive and Developmental Biology, Rm 2006, Department of

10 Surgery and Cancer, Imperial College London, London, W12 0NN, UK

11 Tel: +44(0)20 759 42128

12 Email: [email protected]

13 Abbreviated Title: Spatial control of GPCR signaling

14 Key words: G protein-coupled receptor, trafficking, signaling, microdomain, endosome,

15 reproduction.

16

17 Word Count: 5387

18 Number of Figures: 1

19

20 Disclosure Statement: The authors have nothing to disclose

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21 ABSTRACT

22 Probing the multiplicity of hormone signaling via G protein-coupled receptors (GPCRs) has

23 demonstrated the complex signal pathways that underlie the multiple functions these receptors play in

24 vivo. This is highly pertinent for the GPCRs key in reproduction and pregnancy that are exposed to

25 cyclical and dynamic changes in their extracellular milieu. How such functional pleiotropy in GPCR

26 signaling is translated to specific downstream cellular responses, however, is largely unknown.

27 Emerging data strongly supports mechanisms for a central role of receptor location in signal

28 regulation, via membrane trafficking. In this review, we discuss current progress in our understanding

29 of the role membrane trafficking plays in location control of GPCR signaling, from organized plasma

30 membrane signaling microdomains, potentially provided by both distinct endocytic and exocytic

31 pathways, to more recent evidence for spatial control within the endomembrane system. Application

32 of these emerging mechanisms in their relevance to GPCR activity in physiological and

33 pathophysiological conditions will also be discussed, and in improving therapeutic strategies that

34 exploits these mechanisms in order to program highly regulated and distinct signaling profiles.

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35 INTRODUCTION

36 Within any cellular signaling system it is becoming apparent that signal location is a critical

37 mechanism for cells to translate complex signaling networks at a spatial and temporal level, into

38 specific downstream responses. The membrane trafficking of receptors and the signaling molecules

39 they activate, can direct the location of intracellular signals, and has such been viewed as an

40 integrated cross-regulatory cellular system (1). Thus, membrane trafficking can regulate many

41 fundamental cellular programs and an increasing number of clinical conditions have been reported to

42 result from defects in membrane trafficking pathways or cargo sorting (1,2). The role of location in

43 signal regulation via membrane trafficking is highly pertinent for the signaling activated by the

44 superfamily of G protein-coupled receptors (GPCRs). A well-established contribution that trafficking

45 plays in GPCR signaling is the ability to regulate heterotrimeric G protein signal patterns (3). A

46 simple but dramatic example is the divergent sorting of GPCRs following ligand-induced endocytosis

47 to either recycling or degradative/lysosomal pathways (see Figure 1) a process that essentially

48 produces opposite effects on receptor signaling via its cognate G proteins (3). Furthermore, the

49 potential to reprogram signaling by altering trafficking or location of receptors may be a key adaptive

50 mechanism for GPCRs that are exposed to cyclical and dynamic hormonal environments, such as

51 those receptors central to regulating reproduction and pregnancy. In this review, we will highlight the

52 recent advances in our understanding that membrane trafficking plays in location control of GPCR

53 signaling, focusing where possible, on reproductive-relevant GPCRs, and the potential therapeutic

54 application and exploitation of these findings.

55 CLATHRIN-COATED PITS-MORE THAN JUST A MODE OF ENDOCYTOSIS?

56 The removal of GPCRs from the cell surface, via the process of endocytosis, is well established to

57 regulate receptor signaling by contributing to heterotrimeric G protein-signal desensitzation. Several

58 pathways have been described to regulate receptor-mediated endocytosis, however, clathrin-mediated

59 endocytosis (CME) via clathrin-coated pits (CCPs) is the most extensively characterized. CCPs are

60 formed by the recruitment and ordering of clathrin coat proteins with cargo, accessory proteins and

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61 adaptors (Figure 1, B, reviewed in (4,5)). A new vesicle is then formed following the scission of the

62 CCP from the membrane by the dynamin family of GTPases (6). The widely accepted model for

63 GPCR recruitment to CCPs is that a ligand-activated receptor is first phosphorylated (by a member of

64 the family of the GPCR kinases (GRKs)), which increases the affinity of the cytoplasmic adaptor

65 proteins arrestin 2 and/or 3 to the receptor. Arrestins have the ability to bind clathrin heavy chain,

66 beta(2)adaptin subunit of the clathrin adaptor AP2 and GPCRs, thus facilitating GPCR recruitment

67 and clustering into CCPs for their subsequent internalization (reviewed in (7)). In addition to

68 uncoupling heterotrimeric G protein signaling from the plasma membrane, arrestins also directly

69 activate GPCR signaling independent of their cognate G proteins, by acting as scaffolds for a variety

70 of signaling proteins (Figure 1, C). Distinct GPCRs can vary in how they engage in receptor-mediated

71 endocytosis including; preferential arrestin recruitment, phosphorylation via distinct kinases, ligand-

72 independent internalization and even arrestin- and/or clathrin-independent internalization. This has

73 been extensively reviewed elsewhere (8-11). Instead we will focus on how these pathways, machinery

74 and distinct clathrin-containing plasma membrane domains may enable tight control of cell surface

75 organization as a mechanism to exquisitely regulate .

76

77 The diverse range of cargo and machinery reported to assemble to CCPs has raised the question of

78 whether there is heterogeneity in localization across CCPs for different receptors, and in turn whether

79 this impacts subsequent sorting and signal activity of a receptor. That distinct receptors may be

80 organized within distinct subsets of CCPs was first reported for the β2-adrenergic receptor (β2AR)

81 (12). A role for such specialized spatial organization of CCPs was then demonstrated to dictate its

82 subsequent sorting to recycling or lysosomal pathways (13), as also shown for the purinergic

83 receptors, P2Y(1) and P2Y(12), that identified two distinct CCP populations (14,15) also with

84 functional implications in differential post-endocytic sorting. Mechanistically, the targeting of

85 receptors to these distinct CCPs was dependent on arrestin and phosphorylation, with P2Y(12) and

86 β2ARs recruited in an arrestin and GRK-dependent manner and P2Y(1) in an arrestin-independent yet

87 PKC-dependent manner. An additional GPCR well known to internalize via an arrestin-independent

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88 manner is the mammalian gonadotropin-releasing hormone receptor (GnRHR (16). Although reported

89 to localize to clathrin and caveolae/lipid rafts depending on the cell type (see below), this receptor

90 rapidly recycles following ligand-induced internalization (17), unlike the arrestin-independent

91 P2Y(1), so it is possible there may be further subspecializations of CCPs that is dictated by

92 associating adaptor molecules. The impact of such subspecialization on receptor activity, and

93 consequently its downstream physiological function, may be to function as ‘bona fide’ signalling

94 microdomains, e.g. via arrestin-dependent signal complexes (Figure 1, C). The potential role of CCPs

95 to act as signaling microdomains is highlighted by the ability of GPCRs to regulate their own CCP

96 residency time. For the β2AR this is achieved by interaction with additional protein scaffold proteins,

97 namely postsynaptic density 95/disc large/zonula occludens-1 (PDZ) proteins, that tether GPCRs to

98 cortical actin, and extend the occupancy time of a receptor in a CCP by delaying the recruitment of

99 dynamin (15). However, other receptors such as the mu- delay the scission activity

100 rather than recruitment of dynamin, and in a PDZ-independent manner (18). Functionally delayed

101 residency time of a GPCR in CCPs provides a mechanism to regulate mitogenic signals on a temporal

102 scale via formation of arrestin signaling scaffolds, as suggested by a recent study measuring residency

103 times with distinct agonists of the cannabinoid CB1 receptor (19). The CB1 cannabinoid receptor, as

104 part of the endocanabinoid system, modulates synaptic function, including negative regulation of

105 GABAergic input to GnRH neurons, suppressing GnRH output and fertility (20) and thus such spatio-

106 temporal regulation of CB1 signaling may be key in its role in modulating release of GABA from the

107 presynaptic membrane under physiological or pathophysiological conditions, e.g. the ability of

108 cannabis drugs to inhibit pituitary LH secretion. Additional downstream cellular consequences for

109 tight spatial control of GPCR signalling, such as that generated from CCPs, has been recently

110 demonstrated between PAR2 and neurokinin1 (NK1) receptor whose arrestin-dependent ERK

111 signaling mediates either cell migration (PAR2) or proliferation (NK1 receptor) via differential ERK

112 localization (21). While arrestin-recruitment or post-endocytic trafficking fate of a GPCR could

113 dictate the localization of receptors in to distinct CCPs, clathrin-associated adaptors such as arrestin

114 and AP-2 represent core CCP machinery utilised by many kinds of receptors, but it is uncertain

115 whether there are additional machinery that could direct receptors into distinct pits. It has been

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116 postulated, however, that organization of receptors across CCPs may be initiated via the differential

117 phosphorylation of GPCR intracellular domains (22) although it remains to be determined if this

118 dictates any adaptor protein specificity in addition to the arrestins.

119

120 Lattices and plaques-novel clathrin-containing structures to regulate GPCR signaling in

121 reproduction?

122 Additional plasma membrane clathrin-coated structures, clathrin plaques and clathrin flat lattices,

123 have been recently reported to be potential sites of GPCR organization at the plasma membrane

124 (23,24). Although observed by cell biologists for many years, these recent studies have proposed that

125 they are perhaps unprecedented signal microdomains for GPCRs. These large and stable clathrin-

126 coated structures, with distinct morphologies to CCPs, are present in different cell types, and where

127 GPCRs such as the β2AR, mu-opioid receptor and the chemokine receptor CCR5 can localize. For

128 CCR5, ligand activation specifically reorganized the receptor to flat clathrin lattices. The stable nature

129 of these structures compared to CCPs has led to the suggestion it represents a mechanism to

130 functionally compartmentalize the activated form of the receptor in the plasma membrane and thereby

131 act as a platform for receptor signaling and/or desensitization (24). CCR5 is a key receptor for the

132 immune system and although expressed in leukocytes CCR5 and its ligand are found in other cell

133 types in the reproductive tract where its expression in human endometrium is thought to regulate

134 blastocyst implantation(25). Furthermore presence of the ligand RANTES in follicular fluid and

135 CCR5 on human sperm, its role in chemotaxis of human sperm (26) is of interest in patients with

136 endometriosis and genital tract inflammatory diseases where high levels of RANTES induces a

137 premature acrosome reaction and may underlie the subfertility observed in these patient groups (27).

138 These physiological/pathophysiological roles of CCR5 may reflect the impact of a sustained CCR5

139 signal profile by localization to such plasma membrane coated structures. Interestingly cell adhesion

140 is thought to occur at membrane sites containing these clathrin-coated structures (23,24), and an

141 example where clathrin-coated structures are known to exist at sites of inter-cellular interactions are

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142 tubulobulbar complexes in the testes. Tubulobulbar complexes are elongated tubular extensions that

143 project either from spermatids or, from Sertoli cells into corresponding invaginations of adjacent

144 Sertoli cells at sites of cell/cell junctions (28). Characteristically each membrane complex is capped

145 by a clathrin-coated structure that is highly stable and does not undergo scission; features of the more

146 stable clathrin lattices and plaques rather than the classic CCPs they are proposed to be (28). No

147 GPCRs have been identified to date to be present in these clathrin-coated structures, however, given

148 the expression of the FSH receptor in Sertoli cells and the numerous GPCRs that have been reported

149 to be expressed in sperm (29-31), they could represent an important spatial mechanism in inter-

150 cellular communication for GPCRs and spermatocyte translocation and release.

151

152 Arrestin-dependent signaling and its role in reproduction

153 The ability of arrestin-mediated trafficking to activate cell signaling, in addition to G protein-signal

154 desensitization, has been the topic of extensive study for many years and is of current high interest as

155 a valid pathway for pharmaceutical targeting with clinical benefits (32,33). Arrestins can link a

156 number of effectors to agonist-bound GPCRs, for instance Src family tyrosine kinases, components of

157 the MAP kinase cascade (ERK and JNK) and ubiquitin ligases (Mdm2) (34), giving rise to distinctive

158 temporal and spatially regulated signaling modes induced by GPCRs. These distinct signal modalities

159 provide a mechanism for diversifying signaling via a single class of proteins, and for receptors

160 exposed to dynamic hormonal environments, such as those in reproduction and pregnancy, a way to

161 potentially reprogram GPCR signal pathways as a physiological adaptive mechanism.

162

163 The gonadotropin hormone receptors - follicle-stimulating hormone receptor (FSHR) and luteinizing

164 hormone receptor (LHR) – recruit arrestin for desensitization of gonadotropin-hormone induced

165 signaling but also activation of non-G protein mediated pathways in the ovary and testis. For LHR,

166 the mid-cycle LH surge results in desensitization of Gαs-adenylate cyclase-cAMP signaling in ovarian

167 follicles, a process mediated by arrestin 2 and requiring the ADP-ribosylation factor (ARF)

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168 nucleotide-binding site opener (ARNO). ARNO releases arrestin 2 from its membrane-docking site

169 and a guanine nucleotide exchange factor of the small G-proteins ADP ribosylation factors (Arfs) 6,

170 (ARF6) promotes binding of arrestin to the LHR (35,36). Desensitization of Gαs signaling during the

171 LH surge may ‘bias’ the receptor towards other G protein pathways namely Gαq/11, which is

172 involved in ovulation (37), and/or MAPK pathways, although the involvement of arrestin in these

173 pathways in the ovary remains to be determined. However, arrestin can mediate mitogenic signaling

174 from LHR in testicular Leydig cells, where LHR activates MAPK signaling via an arrestin3/Fyn

175 complex that releases EGF-like factors to activate ERK (38), furthermore this LH and EGF receptor

176 cross-talk is important in driving testosterone production in vivo (39). For FSHR, although it can

177 recruit arrestins and GRKs to desensitize its G protein signaling, it can also induce MAPK signaling

178 via the arrestins. The ability of FSHR to activate MAPK signalling independent of the cAMP pathway

179 likely underlies the subfertility phenotype in an inactivating mutation (A189V) identified in male

180 patients that exhibit impaired cAMP signalling but maintain arrestin-dependent ERK activation (40).

181

182 GPCR signaling is also tightly controlled during pregnancy and undergoes dramatic adaptive changes

183 during labour where (OTR) is central to these processes. Arrestin 2 and 3 in the

184 mouse myometrium negatively regulates OTR-induced contractions, where arrestin2/3 null mice

185 exhibit a lack of OT-induced desensitization (41). Interestingly, in human myocytes, arrestin 2 and 3

186 play contrasting roles in signal desensitization and activation of MAPK signalling. Arrestin 2 is

187 involved in G protein desensitization while arrestin 3 mediates ERK signaling responses (42). Such

188 arrestin-specific regulation may be a mechanism to modulate distinct pro-labor functions of OTR,

189 namely contractions and inflammation (43,44). More recently a central role for arrestins in regulating

190 hypothalamic-pituitary-gonadal function at the level of the receptor has been demonstrated

191 whereby either arrestin 2 or arrestin 3 knockout mouse models exhibited a diminished kisspeptin-

192 dependent LH secretion (45). This suggests that activation of arrestin-mediated ERK signalling by the

193 kisspeptin receptor is an important pathway in reproduction and an explanation why known

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194 inactivating kisspeptin receptor mutations (in terms of Gαq/11 signaling) results in only partial

195 gonadotropic deficiency (45).

196

197 LIPID RAFTS & CAVEOLAE AS GPCR SIGNALING MICRODOMAINS

198 Lipid rafts and caveolae are defined as planar microdomains of the plasma membrane enriched in

199 specific lipids and proteins such as cholesterol, sphingolipids and glycosylphosphatidylinositol (GPI)-

200 anchored proteins or for caveolae, caveolin proteins (Figure 1, Ai). Lipid rafts are known to be

201 involved with various cell functions such as cholesterol homeostasis, protein and lipid sorting and

202 vesicular transport (46,47). Importantly, they are known to regulate cell signaling at the plasma

203 membrane by organizing receptors with its signaling machinery; heterotrimeric G-proteins, effector

204 enzymes such as adenylate cyclase, and regulators of G-protein signaling (e.g. RGS16 (48)). Thus,

205 disruption of lipid raft formation can abolish signaling from certain GPCRs including the NK1

206 receptor (49) and the serotonin 1A receptor (50).

207

208 Localization of GPCRs to lipid rafts can occur in a constitutive or ligand-dependent manner. The

209 majority of GPCRs exhibit a small amount of constitutive partitioning to lipid rafts, which is then

210 increased upon ligand stimulation. However, the murine GnRHR has been shown to exclusively

211 reside in cholesterol-rich membrane microdomains in certain cell types (51,52). Mammalian GnRHRs

212 are unique to the GPCR superfamily by their lack of an intracellular C-terminal tail, however this has

213 been shown to not be the only contributing factor involved in lipid-raft localisation as addition of the

214 chicken GnRHR C-terminal tail had no effect on raft localization (53,54). In contrast, the C-terminal

215 tail of the non-lipid raft associated LHR could reorganize the GnRHR into non-raft regions of the

216 plasma membrane, (53,55), even though LHR has been observed to form ordered plasma membrane

217 microdomains following hormone stimulation (56,57). Interestingly pituitary gonadotropes do not

218 contain caveloae (54) yet the lipid raft protein flottilin-1 is highly expressed and associates with

219 GnRHRs (51), suggesting the nature of the lipid rafts in gonadotropes are distinct. The role of these

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220 microdomains on GnRH-signaling is proposed as a mechanism to compartmentalize calcium signaling

221 via L-type calcium channels in gonadotropes as a required element of GnRH-induced ERK signaling.

222 More recently, total-internal reflection microscopy (TIRF-M) studies in the gonadotrope cell line

223 alphaT3-1, provided direct visual evidence for organised plasma membrane calcium influx events

224 termed ‘calcium sparklets’ (58). Ultimately such spatial control of calcium and ERK signaling by

225 GnRHR may be required to regulate key functions such as LHβ synthesis and secretion (59).

226 Furthermore, they may also represent a platform for receptor cross-talk. Indeed both the

227 glucocorticoid receptor and receptors have been reported to localize to GnRHR-containing

228 lipid raft/microdomains (53,54) For the glucocorticoid receptor, localization to lipids rafts with

229 GnRHR and activation of both receptors was required for the synergistic inhibition of cell

230 proliferation via PKC activation and SGK-1 transcription (60). Interestingly, insulin and GnRHR

231 containing microdomains exhibit asymmetric functionality, whereby insulin enhances GnRH-induced

232 ERK signaling, yet GnRH inhibits activation of insulin-mediated PI3K/Akt signaling pathway (54).

233 This study highlights the important contributions of metabolic pathways in reproduction, and

234 potentially in conditions such as polycystic ovarian syndrome (PCOS) where hyperinsulinaemia could

235 be coupled to the high circulating LH levels found in subsets of women with PCOS (61). However,

236 the majority of such studies are carried out under sustained treatments of GnRH, yet GnRH

237 stimulation is both pulsatile and dynamic, and this has been recently shown to impact on its MAPK

238 signaling (62). Therefore an important next step is to understand how these microdomains contribute

239 to its signaling responses under such dynamic conditions in vivo.

240

241 Despite the caveats involved in the biochemical study of lipid rafts (for review please see (63) the

242 identification that caveolae contain caveolin proteins Cav1, -2, and/or -3 (the latter being muscle-

243 specific) (64), provided a molecular ‘tag’ to study these microdomains. Certain GPCRs have reported

244 to internalize via caveolae, although the molecular detail in how this process occurs is not well

245 understood (65). However, like lipid rafts, they can also act as GPCR signalosomes, both as a means

246 to spatially desensitize and also locally amplify signal responses. Many GPCRs, G-protein subunits

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247 and effector enzyme systems have been shown to be sequestered by caveolae (66,67). One example of

248 a signaling complex mediated by Cav-1 is that of Gαq and phospholipase C, which results in elevated

249 levels of inositol triphosphate (68). Such enhancement of Gαq signaling may underlie the caveolae-

250 dependent anti-proliferative action of OTR in prostatic stromal cells with implications in the

251 dysregulation of this pathway in aging and prostate cancer (69).

252

253 PLASMA MEMBRANE ORGANISATION OF RECYCLED RECEPTORS

254 Following GPCR endocytosis, many receptors are targeted to a recycling pathway back to the plasma

255 membrane, resulting in resensitisation or recovery of hormone signaling (Figure 1, F). GPCRs are

256 sorted to the recycling pathway via a sequence-directed mechanism requiring specific cis-acting

257 sorting sequences in their carboxy-terminal tails (3,70,71). This is in contrast to receptors that recycle

258 in the absence of sorting sequences and occur in a ‘default’ manner via the bulk membrane flow. For

259 some receptors such as the β2-AR, recycling sequences are type 1 PDZ ligands (72,73) and β2AR

260 associates with the PDZ protein SNX27 for its post-endocytic trafficking (74). However the high

261 diversity in recycling sequences across GPCRs suggests they bind specific cytoplasmic proteins (3).

262 We refer the reader to reviews describing the endosomal mechanisms mediating post-endocytic

263 sorting to these pathways via highly diverse GPCR recycling sequences (3,70). In terms of plasma

264 membrane organization of GPCR signaling, recent reports studying the events downstream of

265 endosomal sorting of GPCRs back to the plasma membrane may indicate that such pathways could

266 direct receptors to defined plasma membrane domains. Such studies have employed N-terminal

267 labeling of GPCRs with superecliptic pHluorin (which is fluorescent at neutral pH and non-

268 fluorescent at acidic pH (pH < 6.0) that occurs within the acidic lumen of the endosome), coupled

269 with TIRF-M imaging and has enabled direct visualization of individual receptor insertion events into

270 the plasma membrane at high temporal resolution (71,75,76). The β2AR and mu-opioid receptor both

271 undergo sequence-directed recycling and these studies imaging individual recycling events revealed

272 that GPCR recycling; 1) occurs at a much faster rate following ligand stimulation than previously

273 detected by other approaches and 2) exhibited distinct spatial-temporal patterns of insertion. Both

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274 transient and ‘persistent’ insertion events of receptor containing vesicles have been described, the

275 former remaining for <1 second, while the persistent for >10 seconds before the receptor laterally

276 diffuses in to the membrane. The type of events exhibited seems to be cell-type-dependent as the

277 persistent events were primarily a feature of neurons and not in heterologous cell lines such as HEK

278 293 cells or astrocytes and fibroblasts (75,77). It remains to be determined if other cell types exhibit

279 these kind of persistent fission events and what functional role they may play, although, a role for

280 these events as specialized signaling complexes has been proposed (75). Interestingly, both recycling

281 events seem to be regulated by distinct mechanisms, with the transient events negatively regulated by

282 PKA and agonist, while the persistent is positively regulated by agonist yet PKA-independent. Given

283 the role of GPCR-induced cAMP-PKA signaling in these recycling events, one possible signaling

284 complex involved in regulating and/or perhaps integrating a functional response from these newly

285 recycled receptors are the A-kinase anchoring proteins (AKAPs).

286

287 The AKAPs are a large family of scaffolding proteins that facilitate GPCR signaling by binding to the

288 regulatory subunits of the PKA via their C-terminal motif (reviewed in (78,79)). In addition to the

289 ability of AKAP to interact with PKA, AKAPs are also known to mediate signal transduction by

290 scaffolding signaling machinery such as adenylate cyclase and other kinases, such as serine/threonine

291 and tyrosine kinases, as well as signal termination molecules such as phosphatases and

292 phosphodiesterases. Therefore, AKAP can form specialized signaling microdomains or stations,

293 which can allow spatio-temporal regulation of signaling. AKAPs such as AKAP12 (also termed

294 Gravin or AKAP250) have been shown to preferentially associate with the β2AR at the plasma

295 membrane and are involved in regulating the recycling of this GPCR (80,81). Additional AKAPs

296 implicated in regulation of β2AR receptor activity are AKAP5 (also termed AKAP79/150), which

297 interestingly is specifically required for ERK activation without affecting cAMP signaling or

298 recycling (82). Whether such scaffolding proteins could reflect a potential functional role of persistent

299 and/or transient recycling events remains to be determined. However, AKAP5 and 7alpha have been

300 implicated in maintaining oocyte meiotic arrest, and as both constitutive endocytosis and recycling are

301 required of the GPCRs responsible for maintaining this cAMP signaling (GPR3/6/12), suggests that

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302 spatial scaffolding of cAMP signaling of these recycling GPCRs is important in oocyte maturation

303 (83-85)

304

305 ENDOSOMAL GPCR SIGNALING

306 As described above the paradigmatic model of GPCR signaling used to be depicted as plasma

307 membrane localized receptors activating a distinct heterotrimeric G protein signaling pathway that in

308 turn converges on to common downstream pathways, such as MAPK signaling. Our current

309 understanding of the high complexity in these pathways now include the well-described ability of key

310 GPCR adaptor proteins, i.e. arrestins, in generating distinct signaling patterns at specific membrane

311 locations. However, more recently there has been an emerging body of data demonstrating that G

312 protein signaling from receptors can occur beyond the plasma membrane (i.e. at the level of

313 endosomes) and that signaling could be regulated across endosomal populations, thus GPCR signaling

314 can therefore be broadly categorized in to two phases: plasma membrane signaling (phase 1) and

315 endomembrane signaling (phase 2) (see Figure 1). Pivotal studies demonstrating that GPCRs continue

316 to activate, or exhibit persistent, heterotrimeric G protein signaling following internalization was

317 shown with two Gαs-coupled receptors, the thryotropin-stimulating hormone receptor (TSHR) and

318 the receptor (PTHR). These studies employed cAMP biosensors (Epac1-camps)

319 that demonstrated persistent cAMP signaling requires internalization, the machinery to generate such

320 persistent cAMP signals, Gαs and adenylate cyclases, were localized in early endosomes and that this

321 persistent endosomal signaling is potentially physiologically relevant (as demonstrated for TSHR in

322 3D cultures of thyroid follicles) (86,87). For PTHR, endosomal cAMP generation was shown to be

323 enhanced by arrestin and attenuated by the retromer complex involved in receptor recycling (88) and

324 that endosomal acidification plays a key role in this deactivating switch (89). The PTHR has critical

325 roles in regulating Ca2+ homeostasis by its actions on bone and kidney, and the sustained signalling

326 induced by distinct PTH ligands in turn impacts on trabecular bone volume but induces greater

327 increases in cortical bone turnover. Such features of PTHR signalling may be exploited

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328 therapeutically such as in certain forms of hypoparathyroidism that respond to PTH. However, the

329 authors propose that the PTH analogs that induce sustained signalling lead to enhanced cortical bone

330 turnover, thus conditions such as osteoporosis may not benefit from PTH ligands with sustained

331 signalling properties (90).

332

333 Direct evidence that a GPCR can activate heterotrimeric G proteins from endosomes came from a

334 seminal study that developed a nanobody biosensor to detect either the active, nucleotide free form of

335 Gαs or the ligand-activated conformation of the β2AR (91). These nanobodies were originally

336 generated as tools to aid crystallization of this GPCR in distinct active conformations (92), but the

337 application as a biosensor for imaging in live cells revealed that β2AR and Gαs were reactivated upon

338 reaching the early endosomal membrane (91). The functional role of this endosomal Gαs-cAMP

339 signaling has been recently demonstrated to activate distinct downstream transcriptional responses

340 (93). Interestingly, through the use of optogenetic adenylate cyclase probes targeted to either plasma

341 membrane, endosome or cytoplasm, these transcriptional responses were mediated entirely by the

342 location of the cAMP signal (93), possibly suggesting that endosomally generated cAMP is ‘marked’

343 by as yet unknown mechanisms for the cell to translate these events in to changes in expression of

344 specific genes.

345

346 The spatial organization of second messenger signaling is likely to be important in a wider

347 physiological context and for other GPCRs and G protein pathways. Indeed, sustained endosomal

348 cAMP signaling has been also been reported for the -like 1 receptor (GLP-1R) with a

349 requirement in glucose-stimulated insulin secretion (94) (95), the V2 with roles

350 in regulating renal water and sodium transport and the differential antidiuretic effect between

351 vasopressin and oxytocin (96), the pituitary adenylate cyclase activating polypeptide (PACAP) type 1

352 receptor with roles in PACAP-induced neuronal excitability (95) and potentially the prostaglandin E2

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353 receptor EP4 in order to stimulate production of amyloid-beta that are involved in the

354 pathogenesis of Alzheimer’s Disease (97).

355

356 Endosomal G protein signaling is also relevant for reproductive systems as has been demonstrated for

357 the kisspeptin receptor, a Gαq/11 coupled GPCR that exhibits a persistent calcium signaling profile in

358 CHO cells and the hypothalamic GnRH expressing neuronal cell line GT1-7 (98). This persistent

359 signal was entirely dependent on receptor internalization (98) and may in part underlie the enhanced

360 activity of the R386P kisspeptin receptor mutation, that causes central precocious puberty, due to its

361 decrease in receptor degradation/downregulation and net increase in recycling, and potentially

362 enhanced endosomal calcium signaling (99). Interestingly, this study also demonstrates that

363 endosomal signaling from a GPCR can occur for additional G protein pathways.

364

365 Spatial Regulation of GPCR Signaling Across Divergent Endosomes-the Very Early Endosome

366 Conventionally, the early endosome is viewed as both the earliest and primary platform for receiving,

367 organizing, and sorting diverse sets of cargo, however our recent work has demonstrated that two

368 Gαs-coupled GPCRs, that undergo sequence-directed recycling, the β2AR and the human LHR

369 (rodent LHRs lack the recycling sequence and are primarily sorted for degradation (100) are

370 differentially sorted to distinct endosomal compartments. The β2AR is well known to rapidly

371 internalize and sort from early endosomes, however, the LHR internalized to a physically smaller

372 endosome compartment devoid of early endosomal markers EEA1 and phosphatidylinositol-3

373 phosphate. The endosomal size and biochemical content suggested the LHR internalizes to a distinct

374 compartment upstream of the early endosome (101). It is known that a subpopulation of early

375 endosome intermediates exist that are considered precursors of classic sorting endosomes and notably

376 recruit the effector protein of the GTPase Rab5, APPL1 (Adaptor protein containing PH domain, PTB

377 domain and Leucine zipper motif) (102). Although LHR was found to traffic to APPL-positive

378 endosomes, this receptor does not require Rab5 for its activity suggesting that the pre-early

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379 endosomes that LHR traffics to may be a distinct compartment (101). Routing of the LHR to this

380 smaller pre-early endosomal compartment was dependent on the receptor interacting with the PDZ

381 protein Gαi-interacting protein C-terminus (GIPC), which was previously shown to interact with the

382 LHR C-tail and required for its recycling (103). These studies are complementary as a loss of this

383 interaction inhibits LHR recycling due to the rerouting of this receptor away from pre-early

384 endosomes to early endosomes. This indicated a sorting requirement for this compartment and hence

385 may represent a very early endosome (VEE) (Figure 1, D). It also demonstrated that sequence-

386 directed sorting of GPCRs can occur from compartments other than the early endosome, as has also

387 been demonstrated recently for the β1-adrenergic receptor (β1AR) (104).

388

389 The very early endosome also provided a mechanism for direct spatial control of GPCR signaling.

390 LH-induced activation of the LHR induced a sustained temporal profile of ERK signaling that

391 required both internalization and targeting to the correct endosomal compartment, the very early

392 endosome (101). Thus, in addition to the compartmental bias in heterotrimeric G protein signaling

393 between the plasma membrane and early endosomes described in the studies above, there is also

394 compartmental bias in GPCR signaling across distinct endosomes. APPL endosomes have been

395 shown to act as signaling centers to regulate the MAPK and Akt signaling cascades by EGFR,

396 and TrkA receptors and could be crucial for cell survival in some systems (105-109). For

397 LHR, the downstream role of very early endosomal targeting remains to be determined, however LH

398 does induce a sustained ERK signaling profile in human granulosa cells that negatively regulates

399 aromatase expression (110), a signaling profile that may result from localization to and trafficking

400 from very early endosomes. However, the known hyperandrogenemia that is one of the diagnostic

401 hallmarks of PCOS may be a result of altered LHR endosomal location or retention in very early

402 endosomes that could result in altered or enhanced signal profile leading to the known increased

403 androstenedione production by LHR in theca cells(111,112);.

404

16

405 The very early endosome may be key in regulating activity for GPCRs in addition to the LHR, as has

406 been shown for the β1AR and the FSHR (101). Although the β1AR is a known GIPC-interacting

407 GPCR (113), the requirement of GIPC in FSHR signaling has not been reported, although APPL1 is a

408 well-known binding partner of FSHR (114) and APPL1 and GIPC can also associate (108) suggesting

409 a possible mechanism of very early endosomal regulation by these GPCRs. Overall the finding that

410 distinct endosomes can confer defined spatial control of receptor signaling provides a cell system

411 where hormonal signaling could be altered by simply rerouting receptors between compartments

412 under both physiological and pathophysiological conditions.

413

414 CONCLUDING REMARKS AND FUTURE DIRECTIONS

415 Spatial control of GPCR signaling is more than just an emerging concept but now a working cellular

416 model (Figure 1) that now can be employed to understand how cells translate complex signaling

417 responses into defined cellular programs in vivo. However, the molecular details of these pathways for

418 a given receptor are likely to be even more complex. A detailed understanding of these fundamental

419 cell biological mechanisms will likely accelerate with advances in imaging technology such as the

420 broad spectrum of super-resolution microscopy approaches that are currently being employed,

421 enabling visualization of proteins below the diffraction barrier of standard immunofluorescence

422 microscopy, including TIRF-M, and even its adaptation to studying molecular processes directly in

423 vivo (115).

424

425 GPCRs remain one of the most successful drug targets, and harnessing or manipulating the spatial

426 control of GPCR signaling is already being targeted for novel therapeutic strategies. Biased ligands

427 are of current high interest with some in clinical development (116,117). Such compounds can

428 stabilize a subset of multiple possible receptor conformations, leading to high selectivity in its action

429 and potentially fewer off target effects. These ligands can target either orthosteric or allosteric binding

17

430 sites in the receptor, or even intracellular regions of the receptor as shown recently through the use of

431 intrabodies or pepducins (120-123). Currently this pharmacological ‘tool box’ targets signal bias

432 between distinct G protein pathways or, between G protein and arrestin-mediated signaling. As the

433 mechanisms and physiological roles of spatial control of GPCR signaling are understood, current

434 compounds could be further refined, to accommodate the new information on these pathways and/or

435 represent a platform to screen for novel ligands with highly defined properties in directing receptor

436 location and thus spatial changes in GPCR signaling. Furthermore, endogenous hormones could be

437 biased ligands, such as the distinct glycosylation variants of hCG and FSH that are known to have

438 distinct biological activities (118,119). In terms of pharmaceutical development in targeting

439 reproductive-relevant receptors, a number of small molecule allosteric compounds to GnRHR, LHR

440 and FSHR have been developed (124,125). While the pharmacochaperone properties of some of these

441 compounds, such as those to GnRHR, have been well described (126), the role of these small

442 molecules in spatial control or biased signaling remains to be determined. In addition to drug design,

443 the approach that is used to characterize GPCR mutations or polymorphisms must also be revised to

444 incorporate these latest models of GPCR signaling, such as has been demonstrated with the A189V

445 FSHR mutation (40) and the R386P kisspeptin receptor mutation, that causes central precocious

446 puberty (99).

447

448 In summary, spatial organization and thus the cellular location of a GPCR, can program the

449 multidimensionality of GPCR signaling into highly regulated and distinct signaling profiles, a

450 property that is highly advantageous for dynamic hormone signaling. Efforts must now be directed to

451 exploit this new information in order to provide important insight in to complex diseases and

452 conditions such as PCOS, gonadotropin-dependent cancers and pre-term labour, and improving

453 therapeutic strategies that incorporates the full pleiotropic nature of these receptors.

454

455

18

456 Acknowledgments

457 This work was supported by grants from Genesis Research Trust.

458 Abbreviations:

459 AKAP A kinase anchoring protein

460 APPL1 Adaptor protein containing PH domain, PTB domain and Leucine zipper motif

461 ΑRΝΟ ADP-ribosylation factor nucleotide-binding site opener

462 β1AR β1-adrenergic receptor

463 β2AR β2-adrenergic receptor

464 CCP clathrin-coated pit

465 CME clathrin-mediated endocytosis

466 FSHR follicle-stimulated hormone receptor

467 GIPC Gai-interacting protein, C-terminus

468 GPCR G protein-coupled receptor

469 GnRHR gonadotropin-releasing hormone receptor

470 GRKs GPCR kinases

471 LHR luteinizing hormone receptor

472 MAPK mitogen activated protein kinase

473 NK1 neurokinin-1

19

474 OTR oxytocin receptor

475 PACAP pituitary adenylate cyclase activating polypeptide

476 PCOS polycystic ovarian syndrome

477 PDZ postsynaptic density 95/disc large/zonula occludens-1

478 PGE2 prostaglandin E2

479 PTHR parathyroid hormone receptor

480 TIRF-M total-internal reflection microscopy

481 TSHR thyrotropin-stimulating hormone receptor

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846

847

33

848 FIGURE LEGEND

849 Figure 1. A cellular model for spatial control of GPCR signaling across the plasma membrane

850 and endomembrane system. Following agonist binding, GPCRs can be directed into distinct plasma

851 membrane microdomains such as lipid rafts (Ai) or, following activation and subsequent

852 desensitisation (Aii), several GPCRs are known to undergo endocytosis via clathrin-dependent or

853 independent mechanisms (B). Clathrin-coated pits (CCPs) allow accumulation of GPCR/arrestin

854 complexes that can activate arrestin-mediated mitogenic signaling (C). Following internalization,

855 GPCRs will accumulate in endosomal compartments where they can continue or reactivate

856 heterotrimeric G protein signaling from the endosomal membrane, in addition to scaffolding arrestin

857 to activate G protein-independent signal responses (D & E). Some GPCRs are differentially sorted to

858 a distinct endosomal population termed pre-EEs or very early endosomes (VEEs) (D), via recruitment

859 and binding of the PDZ protein GIPC at the CCP. The VEE sorts receptors to the regulated, sequence-

860 directed plasma membrane recycling pathway and targeting of GPCRs to this compartment generates

861 a sustained MAPK signaling response, although it is unknown if other pathways, including G protein

862 signaling are activated in this compartment. Other GPCRs are directly routed to the early endosome

863 (EE), where they are differentially sorted between recycling and degradative/lysosomal pathways,

864 leading to heterotrimeric G protein plasma membrane signal resensitization or signal termination

865 respectively. Reinsertion in to the plasma membrane of GPCRs targeted to the recycling pathway (F)

866 exhibits distinct spatial-temporal patterns of insertion, either transient or persistent (see text), although

867 its role in spatial control of signaling in addition to resensitization is unknown.

868

869

34

B AiAi AiiAii DESENSITIZED Lipid raft Clathrin- Caveolin- Clathrin- and RESENSITIZED Cholesterol dependent dependent caveolin- endocytosis endocytosis independent

G-GTP G-GTP Glycolipid Phase 1:

G-protein P Plasma G-protein C Arrestin mediated mediated Persistent Transient membrane D Bulk GIPC signalling APPL1 recycling F VEE Sequence- dependent recycling MAPK Other pathways?

EEA1

EE Phase 2: Endomembrane signalling

G-GTP MVB Arrestin P E G-protein Lysosome mediated MAPK Other pathways