To determine whether heterologous expression could be used to identify GPCR ciliary localization sequences, we generated constructs encoding mouse somatostatin receptors one through five (Sstr1–5) fused at the C-terminus to enhanced green fluorescent protein (EGFP). These constructs were then expressed in inner medullary collecting duct (IMCD) cells, which are derived from mouse kidney and develop cilia in culture. Visualization of the subcellular localization of the fluorescently labeled receptors 24–48 h after transfection showed that of the somatostatin receptor subtypes, only Sstr3 selectively localized to cilia (, A–E). The remaining somatostatin receptor subtypes failed to localize to cilia, as indicated by a lack of colocalization with the ciliary marker acetylated α-tubulin, and instead localized to the plasma membrane or within intracellular compartments. Similarly, transfection of IMCD cells with Htr6 and the closely related Htr7 fused at the C-terminus to EGFP revealed that Htr6 preferentially localized to cilia, whereas Htr7 localized primarily to the plasma membrane (, F and G). Interestingly, cilia on cells expressing Sstr3 or Htr6 consistently appeared longer than cilia on untransfected cells or on cells expressing nonciliary GPCRs (, A–G). The underlying mechanism for this difference is unknown. Overall, these results suggest that Sstr3 and Htr6 contain sequences that specify ciliary localization, and IMCD cells possess the necessary machinery to traffic specific receptors to cilia.
Figure 1. Somatostatin receptor subtype 3 and serotonin receptor subtype 6 selectively localize to cilia when heterologously expressed in IMCD cells. (A–G) Representative confocal microscopy images of transiently transfected IMCD cells expressing somatostatin (more ...)
To identify the region of Sstr3 containing ciliary localization sequences, we utilized a fusion PCR approach to construct EGFP-fused chimeric receptors. Chimeras were generated containing segments of Sstr3 and the structurally and pharmacologically similar Sstr5. To minimize the likelihood of protein misfolding, the chimeric receptor fusion sites were engineered at conserved residues located within the transmembrane domains. Initially, we generated chimeric receptors of Sstr5 in which the sequence from the N-terminus to the second, fourth, or sixth transmembrane (TM) domains was substituted with the corresponding sequence from Sstr3 (A). Expression of these chimeric receptors in IMCD cells revealed that only chimeric receptor Sstr5[N-TM6Sstr3] selectively localized to cilia (, B–D), suggesting that sequences between the TM4 and TM6 domains in Sstr3 mediate ciliary localization. To test this hypothesis, we generated a chimeric receptor of Sstr5 in which the sequence between the TM4 and TM6 domains was replaced with the corresponding sequence from Sstr3 (A). This chimera also localized to cilia (B), suggesting that ciliary localization sequences are located within the third extracellular (e3) loop or the third intracellular (i3) loop of Sstr3. To distinguish between these two possibilities, we generated chimeric receptors of Sstr5 containing only the e3 or i3 loop of Sstr3 (A). Notably, chimeric receptor Sstr5[TM4-5Sstr3] did not localize to cilia (C), but chimeric receptor Sstr5[TM5-6Sstr3] did localize to cilia (D), indicating that sequences within the i3 loop of Sstr3 are sufficient to localize Sstr5 to cilia. Immunoblotting of proteins isolated from cells transiently transfected with chimeric receptors Sstr5[TM4-5Sstr3] and Sstr5[TM5-6Sstr3] revealed similar expression patterns (Supplementary Figure 1), indicating that absence of cilia localization was not due to the lack of receptor expression or stability.
Figure 2. Sequences between the fourth and sixth transmembrane domains of Sstr3 are important for ciliary localization. (A) Schematic of chimeric receptors containing portions of Sstr3 (indicated by black lines) and Sstr5 (indicated by white lines) fused at the (more ...)
Figure 3. The third intracellular (i3) loop of Sstr3 is sufficient to localize Sstr5 to cilia. (A) Schematic of chimeric receptors containing portions of Sstr3 (black lines) and Sstr5 (white lines) fused at the C-terminus to EGFP. TM domains are depicted as boxes. (more ...)
To determine whether the i3 loop of Htr6 also mediates ciliary localization, we generated a chimeric receptor in which the i3 loop of Htr7 had been substituted with the i3 loop of Htr6 (A). Notably, this chimeric receptor selectively localized to cilia (B). This result indicates that Sstr3 and Htr6 contain ciliary localization sequences within the same domain and suggest that they may be targeted to cilia through similar mechanisms. Because the predicted i3 loop of Htr6 (63 residues) is significantly larger than the i3 loop of Sstr3 (36 residues), we further narrowed the region of Htr6 sufficient to traffic Htr7 to the cilium by generating chimeric receptors containing only the N- or C-portion of the i3 loop (A). V241 in Htr6, which corresponds to V295 in Htr7, was used as the fusion site. Chimeric receptor Htr7[TM5-V241Htr6] selectively localized to cilia (C) but chimeric receptor Htr7[V241-TM6Htr6] did not (D), indicating that sequences within the N-portion of the i3 loop of Htr6 are sufficient to localize Htr7 to cilia.
Figure 4. The amino portion of the i3 loop of Htr6 is sufficient to localize Htr7 to cilia. (A) Schematic of chimeric receptors containing portions of Htr6 (black lines) and Htr7 (white lines) fused at the C-terminus to EGFP. TM domains are depicted as boxes. (B–D) (more ...)
We hypothesized that ciliary localization sequences would be unique to the receptor subtype that localizes to cilia, would be conserved in species in which the receptor is ciliary, and would be similar between Sstr3 and Htr6. Comparison of the i3 loop sequences of the mouse somatostatin receptors reveals similarity between Sstr3 and the other subtypes, except for several residues in the N-portion and an insertion of 11 amino acids that is unique to Sstr3 (A). To further narrow the sequences of interest we compared the i3 loop sequences of Sstr3 across species. Comparison of the mouse and human sequences reveals that the unique residues in the N-portion of the loop are not conserved, but 5 of the 11 amino acids inserted in mouse Sstr3 (APSCQ) are completely conserved in human SSTR3 (B). Given that human SSTR3 also localizes to cilia when expressed in IMCD cells (data not shown), this suggested that residues within “APSCQ” might confer ciliary localization.
Figure 5. Comparative genomics and mutational analysis identifies unique residues in Sstr3 and Htr6 that are important for ciliary localization of GPCRs. (A) Alignment of the predicted i3 loop sequences of the mouse somatostatin receptors reveals an insertion of (more ...)
We then examined the N-portion of the i3 loop of Htr6 and identified a sequence (ATAGQ) with modest similarity that is not present in Htr7 (C). Because the A and Q at the ends of the sequence are identical between Sstr3 and Htr6, we performed site-directed mutagenesis on these residues to verify they are important for ciliary localization. The A and Q residues in chimeric receptor Sstr5[TM5-6Sstr3] were mutated to phenylalanine, and ciliary localization was then quantified in IMCD cells expressing Sstr3, Sstr5, Sstr5[TM5-6Sstr3], or Sstr5[TM5-6Sstr3mut]. Sstr3 localized to cilia in ~91% of transfected cells, whereas Sstr5 never localized to cilia (D). Chimeric receptor Sstr5[TM5-6Sstr3] showed a similar percentage of cilia localization (~93%) as Sstr3 (D and Supplementary Figure 2A). The mutated version of this chimera (Sstr5[TM5-6Sstr3mut1]) showed a dramatic reduction in the percentage of ciliary localization but still localized to cilia in ~50% of cells (D). However, the i3 loop of mouse Sstr3 contains a second ciliary localization consensus sequence (APACQ; B). To test whether the second site was contributing to ciliary localization, we mutated the A and Q residues to F in Sstr5[TM5-6Sstr3mut1] and quantified ciliary localization. Notably, when all four residues were mutated in this chimera (Sstr5[TM5-6Sstr3mut2]) ciliary localization was reduced to ~6% of cells (D and Supplementary Figure 2B). Immunoblotting of proteins isolated from transiently transfected cells expressing chimera Sstr5[TM5-6Sstr3mut2] confirmed that the mutant chimeric receptor was expressed (Supplementary Figure 1).
Similarly, the A and Q residues in chimeric receptor Htr7 [TM5-V241Htr6] were mutated to phenylalanine and ciliary localization was then quantified in IMCD cells expressing Htr6, Htr7, Htr7[TM5-V241Htr6], or Htr7[TM5-V241Htr6mut]. Htr6 localized to cilia in ~93% of transfected cells, whereas Htr7 localized to cilia in ~20% of transfected cells (E). Of note, ciliary localization of Htr7 was almost exclusively seen in cells that were expressing the receptor at a very high level. Chimeric receptor Htr7[TM5-V241Htr6] localized to cilia in ~70% of transfected cells (E and Supplementary Figure 2C). Remarkably, the mutated version of this chimera (Htr7[TM5-V241Htr6mut]) almost never localized to cilia (~4%; E and Supplementary Figure 2D). Together, these results indicate that the A and Q residues in the i3 loop of Sstr3 and Htr6 are important for ciliary localization.
To test whether these sequences could be used to predict novel ciliary GPCRs, we formulated a consensus sequence based on Sstr3 and Htr6. Because serine and alanine belong to a “strong” Gonnet Pam250 matrix conservation group (Gonnet et al., 1992
), we formulated the following loose consensus sequence (AX[S/A]XQ), where position 1 is an A, position 5 is a Q, position 3 is an S or A, and positions 2 and 4 are any amino acid. As the ciliary localization sequences were identified within the i3 loop of both Sstr3 and Htr6, we searched for the consensus sequence in a database containing the sequences of the predicted i3 loop of all human GPCRs. The consensus sequence is present in the i3 loop of 11 additional GPCRs (). Remarkably, these 11 GPCRs include 3 odorant receptors, 3 cone opsins, and rhodopsin, all of which are ciliary proteins. The remaining four GPCRs (α-2A adrenergic receptor, chemokine orphan receptor 1, melanin-concentrating hormone receptor 1, and muscarinic acetylcholine receptor M5) have not been shown to localize to cilia and were classified as candidate ciliary GPCRs. Because melanin-concentrating hormone receptor 1 (Mchr1) is involved in the regulation of feeding behavior and energy balance (Pissios et al., 2006
) and multiple human ciliary disorders are associated with obesity (Badano et al., 2006
), Mchr1 was considered a particularly good candidate ciliary GPCR.
G protein–coupled receptors identified by the ciliary localization consensus sequence
To test whether Mchr1 localizes to cilia, we cloned the coding sequence of Mchr1 from mouse cDNA, fused it to EGFP, expressed it in IMCD cells, and assessed subcellular localization. Notably, Mchr1 localized to cilia when expressed in IMCD cells (A). We then tested whether Mchr1 localizes to cilia in tissue by labeling mouse brain sections with an antibody to Mchr1. We detected ciliary localization of Mchr1 in several brain regions (B), including the hippocampus, nucleus accumbens, olfactory bulb, and hypothalamus. Ciliary localization was confirmed by colabeling with ACIII, which is a marker of neuronal cilia (Berbari et al., 2007
; Bishop et al., 2007
). These results indicate, for the first time, that Mchr1 localizes to cilia in vitro and in vivo.
Figure 6. Melanin-concentrating hormone receptor 1 (Mchr1) localizes to cilia in vitro and in vivo. (A) Representative image of transiently transfected IMCD cells expressing Mchr1 fused at the C-terminus to EGFP. Left, EGFP fluorescence (green); middle, acetylated (more ...)