1 Dopamine receptors reveal an essential role of IFT-B, 2 KIF17, and Rab23 in delivering specific receptors to 3 primary cilia. 4 5 6 7 1,2 1,3,4 8 Alison Leaf and Mark von Zastrow 9 1 2 3 10 Program in Cell Biology, Department of Biochemistry and Biophysics, Department of 4 11 Psychiatry, and Department of Cellular and Molecular Pharmacology, University of 12 California, San Francisco, CA 94158 13 14 15 16 17 18 Running Title: Ciliary targeting of dopamine receptors 19 20 21 22 Address correspondence to: Mark von Zastrow, MC 2140, 600 16th Street, 23 San Francisco, CA 94158-2140. Fax: 415-514-0169 E-mail: [email protected] 24 25 26 Keywords: Cilia, sorting, G protein-coupled receptor, Rab23, signaling, KIF17, 27 intraflagellar transport 28 29 30 31 32 33 Abstract 34 35 Appropriate physiological signaling by primary cilia depends on the specific targeting of 36 particular receptors to the ciliary membrane, but how this occurs remains poorly 37 understood. Here we show that D1-type dopaminergic receptors are delivered to cilia 38 from the extra-ciliary plasma membrane by a mechanism requiring the receptor 39 cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. 40 This targeting mechanism critically depends on Rab23, a small GTP-binding protein that 41 has important effects on physiological signaling from cilia but was not known previously 42 to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine 43 receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non- 44 ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the 45 ciliary membrane. Dopamine receptors thus reveal a previously unrecognized 46 mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this 47 process. 2 48 Introduction 49 50 Primary cilia are microtubule-based protrusions of the plasma membrane that support a 51 wide range of specialized receptor-mediated signaling functions. Physiological signaling 52 from cilia critically depends on the selectivity of receptor targeting to the ciliary 53 membrane, and disturbances in this targeting are thought to underlie a variety of 54 pathological conditions (Hsiao et al., 2012). The remarkable specificity of ciliary 55 membrane targeting is clear among G protein-coupled receptors (GPCRs). Some 56 members of this large receptor family robustly accumulate in the ciliary membrane while 57 others, including closely related homologues, are found throughout the extra-ciliary 58 plasma membrane but are effectively excluded from cilia (Marley and von Zastrow, 59 2010; Schulz et al., 2000). Understanding how particular GPCRs localize to primary cilia 60 with such exquisite selectivity is a fundamental problem with broad physiological 61 significance (Emmer et al., 2010). 62 63 The ciliary membrane compartment is separated from the surrounding extra-ciliary 64 plasma membrane by a transition zone complex that impedes lateral exchange of 65 membrane proteins (Chih et al., 2011; Gilula and Satir, 1972; Hu et al., 2010; Williams 66 et al., 2011). This can explain how GPCRs are retained in cilia once delivered, but not 67 how they are delivered in the first place. Two basic routes of ciliary membrane delivery 68 have been described: First, receptors can originate from an intracellular source, through 69 fusion of post-Golgi transport vesicles with the ciliary membrane in or near the transition 70 zone. A number of membrane proteins are targeted to cilia by this route, and molecular 71 machineries supporting it have been identified (Deretic and Papermaster, 1991; Geng et 3 72 al., 2006; Mazelova et al., 2009). Second, receptors can originate from the extra-ciliary 73 plasma membrane. This route, first described in a seminal study of flagellar agglutinins 74 in Chalmydomonas (Hunnicutt et al., 1990), contributes to ciliary targeting of the atypical 75 seven-transmembrane protein Smoothened (Smo; Milenkovic et al., 2009) in 76 mammalian cells. Is the lateral delivery route relevant to ciliary localization of 77 conventional GPCRs? 78 79 Molecular mechanisms that underlie specific ciliary delivery pathways also remain 80 incompletely understood. A number of proteins are already known to play a role, 81 including the BBSome (Jin et al., 2010; Nachury et al., 2007; Berbari et al., 2008b), 82 Tulp3 (Mukhopadhyay et al., 2010, 2013), Arf4 (Deretic et al., 2005), ASAP1 (Wang et 83 al., 2012) and IFT-B and IFT-A (Mukhopadhyay et al., 2010; Crouse et al., 2014; Keady 84 et al., 2011; Kuzhandaivel et al., 2014; Keady et al., 2012). Are there additional 85 machineries not yet identified that function in targeting specific GPCRs to cilia? 86 87 We addressed these questions through study of the D1-type dopamine receptor (D1R), 88 a conventional GPCR that robustly localizes to cilia in diverse cell types (Domire et al., 89 2011; Marley and von Zastrow, 2010). Here we show that D1Rs are delivered to the 90 cilium from the extra-ciliary plasma membrane. Further, we show that ciliary D1R 91 cytoplasmic tail is both necessary and sufficient to direct receptor targeting to the ciliary 92 membrane, and this requires a distinct set of cellular proteins including the anterograde 93 intraflagellar transport (IFT-B) complex and ciliary kinesin, KIF17. Moreover, we identify 94 an essential role of the small GTP-binding protein, Rab23, in the ciliary targeting 4 95 mechanism. Rab23 is not only necessary for D1R access to cilia, it is also sufficient to 96 drive strong ciliary localization of a non-ciliary GPCR. D1Rs thus reveal a discrete route 97 and mechanism of ciliary GPCR targeting, in which Rab23 plays an unprecedented and 98 essential role. 99 5 100 Results 101 102 D1Rs are robustly targeted to the primary cilium 103 The D1R is a cilia-localized GPCR whose mechanism of targeting to the cilium is poorly 104 understood (Marley and von Zastrow, 2010; Domire et al., 2011; Zhang et al., 2013). We 105 investigated this question using recombinant receptors expressed in inner medullary 106 collecting duct (IMCD3) cells. Using an N-terminal Flag tag on the D1R to label the 107 overall surface pool, D1Rs were visualized throughout the plasma membrane and highly 108 enriched in cilia marked by acetylated tubulin (AcTub) (Figure 1A), like the cilia-localized 109 somatostatin-3 receptor (SSTR3) (Figure 1B; Berbari et al., 2008a; Händel et al., 1999; 110 Schulz et al., 2000). In contrast, the delta opioid peptide receptor (DOP-R or DOR) 111 localized throughout the extra-ciliary plasma membrane, but was not detectable on cilia 112 (Figure 1C). 113 114 We first quantified ciliary localization by counting the number of receptor-expressing 115 cells with visible receptor immunoreactivity on the cilium. This normative metric verified 116 ubiquitous D1R localization to cilia, similar to SSTR3, and high specificity of ciliary 117 localization relative to DOR (Figure 1D). Second, because cilia scored as receptor- 118 positive varied in degree of apparent receptor concentration, we determined average 119 fold-enrichment of receptors on the cilium relative to the extra-ciliary plasma membrane 120 (Figure 1E). This graded metric further verified robust ciliary localization of the D1R and 121 SSTR3 (but not DOR), and indicated that the D1R is enriched on cilia even more 122 strongly than the SSTR3 (Figure 1F). 6 123 D1Rs are mobile in the ciliary membrane and accumulated by continuous delivery 124 from the extra-ciliary plasma membrane pool 125 In principle, D1Rs could be concentrated on cilia relative to the extra-ciliary plasma 126 membrane by immobilization or by a diffusion barrier at the base of the cilium (Francis 127 et al., 2011; Hu et al., 2010; Huang et al., 2007). To distinguish these possibilities, we 128 fused the D1R to photoactivatable GFP (Flag-D1-PAGFP) and investigated mobility by 129 live cell imaging coupled to local photoactivation (Figure 2A). We verified appropriate 130 ciliary localization of engineered receptors by anti-Flag Alexa555 surface labeling 131 (Figure 2B, top row). Locally photoactivated D1Rs were distributed non-uniformly on 132 cilia immediately after the 405 nm illumination pulse and then equilibrated throughout 133 the cilium within seconds (Figure 2B,C; whole cell images shown in Figure 2—figure 134 supplement 1). However, there was not visible spread of labeled receptors outside of 135 the cilium on this time scale. Consistent with this, total PA-GFP fluorescence intensity 136 integrated over the ciliary length remained unchanged throughout an 80 sec interval 137 after local photoactivation (Figure 2D). Further, a robust fluorescence signal 138 representing photoactivated D1Rs was still visible when the same cilia were re-imaged 139 minutes thereafter (Figure 2E; whole cell images shown in Figure 2—figure supplement 140 2). Together, these observations indicate that ciliary D1Rs are laterally mobile in the 141 ciliary membrane compartment, but restricted from freely diffusing into the extra-ciliary 142 plasma membrane. 143 144 Achieving net ciliary concentration of receptors that are laterally mobile requires the 145 ability of new receptors to enter the cilium. We sought to measure this delivery by taking 7 146 advantage of the irreversible nature of PA-GFP photoactivation (Lippincott-Schwartz et 147 al., 2003). Multiple 405 nm light pulses were delivered in rapid succession to 148 photoactivate the majority of D1Rs present in the cilium. We then assessed the effect of 149 administering a subsequent 405 nm pulse at a later time. We reasoned that, because 150 D1Rs delivered from outside the cilium would not have been previously photoactivated, 151 their arrival in the cilium would result in an increment in the ciliary PA-GFP signal elicited 152 by a subsequent 405 nm pulse. This was indeed the case: When a subsequent 405 nm 153 pulse was administered shortly (~ 30 sec) after the initial photoactivation series, little 154 (~15%) increase of ciliary PA-GFP fluorescence was observed, consistent with a small 155 residual fraction of D1Rs escaping photoactivation in the initial pulse series (Figure 2F, 156 left bar).