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Originally published as MBC in Press, 10.1091/mbc.E07-03-0239 on June 20, 2007

Vol. 18, Issue 9, 3277-3289, September 2007

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STAM and Hrs Down-Regulate Ciliary TRP Receptors

Jinghua Hu*, Samuel G. Wittekind{dagger}, and Maureen M. Barr*,{ddagger}

*Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, Madison, WI 53705; and {dagger}University of Washington School of Medicine, Seattle, WA 98195-6340

Submitted March 14, 2007; Revised May 18, 2007; Accepted June 13, 2007
Monitoring Editor: Jean Gruenberg


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of Caenorhabditis elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which down-regulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cilia are specialized organelles that function in motility (motile or nodal cilia) or sensation (sensory or primary cilia). Several human genetic diseases are linked to defects in cilia formation or function (Pazour and Rosenbaum, 2002Go; Badano et al., 2006Go). Ciliary assembly via intraflagellar transport (IFT) and sensory transduction capabilities are evolutionarily conserved (Rosenbaum and Witman, 2002Go). These sensory devices, recently referred to as "antennae" or "nanomachines," transduce a plethora of sensory stimuli and must be fine-tuned both temporally and spatially to execute their cellular functions (Marshall and Nonaka, 2006Go; Scholey and Anderson, 2006Go; Singla and Reiter, 2006Go). Significant advances have been made in understanding cilia biogenesis and the genetic basis of human ciliary disease. In contrast, little is known regarding how cilia perceive, integrate, and transduce multiple extracellular stimuli into precise developmental and physiological responses.

Sensory cilia are best known for their roles in photoreception and olfaction, which require G protein–coupled receptors (GPCRs) on the ciliary membrane (Buck and Axel, 1991Go; Marszalek et al., 2000Go). Cilia also act in mechanosensory and osmotic capacities and require ciliary localization of transient receptor potential (TRP) ion channels (Tobin et al., 2002Go; Kim et al., 2003Go; Nauli et al., 2003Go). Recently, vertebrate cilia have been shown to mediate not only environmental inputs, but also the Hedgehog (Hh) developmental cue that triggers translocation of the Smoothened (Smo) GPCR into the cilium (May et al., 2005Go; Huangfu and Anderson, 2006Go). Vertebrate cilia also express the somatostatin receptor sst3, serotonin 5-HT6 receptor, platelet-derived growth factor receptor {alpha} (PDGFR {alpha}) and epidermal growth factor receptor (EGFR; Brailov et al., 2000Go; Whitfield, 2004Go; Ma et al., 2005Go; Schneider et al., 2005Go). Clearly, receptors and channels must be precisely located and regulated to endow an individual cilium with its specific properties. The mechanisms regulating ciliary membrane localization are not well understood and are likely to involve targeting, trafficking, retention, and endocytic removal. Improper ciliary receptor or channel localization could potentially cause sensory transduction defects and human ciliary diseases.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic disease and a major cause of end-stage renal disease (Igarashi and Somlo, 2002Go). Mutation in either the PKD1 or PKD2 gene is responsible for nearly all ADPKD cases. PKD1 and PKD2 gene products, PC1 and PC2, are members of the transient receptor protein polycystin (TRPP) family of TRP channels (Mochizuki et al., 1996Go) and act as a nonselective cation channel (reviewed in Delmas, 2004Go; Igarashi and Somlo, 2002Go). TRP channels function in a broad range of sensory modalities (Clapham, 2003Go). PC1 and PC2 function together as a mechanosensory receptor/channel complex on kidney primary cilia and may be involved in sensing liquid shear stress and urine flow (Igarashi and Somlo, 2002Go; Pazour et al., 2002Go; Yoder et al., 2002Go; Nauli and Zhou, 2004Go; Delmas, 2005Go).

PKD1 and PKD2 are the founding members of the polycystin gene family. In mammals, five PC1 genes (PKD1, PKD1L1, PKD1L2, PKD1L3, and PKDREJ) and three PC2 genes (PKD2, PKD2L1, and PKD2L2) have been identified. Interestingly, the PC1-PC2 gene family acts as a functional unit in numerous developmental and physiological processes, including mammalian kidney tubulogenesis (PC1 and PC2), fertilization in mammals and sea urchin (PKDREJ and several PC2 family members), mammalian sour taste reception (PKD1L3 and PKD2L1), and Caenorhabditis elegans mating behavior (LOV-1 and PKD-2; Barr et al., 2001Go; Neill et al., 2004Go; Delmas, 2005Go; Ishimaru et al., 2006Go; Sutton et al., 2006Go). The polycystins have been found in various subcellular compartments, including the endoplasmic reticulum (ER), plasma membrane (PM), and cilium. The knowledge of polycystin localization mechanisms is critically important for understanding their roles in health and human disease.

The transparent nematode C. elegans provides a powerful model to study human ciliary diseases, ciliogenesis, sensory transduction, and ciliary receptor localization in vivo (Barr, 2005Go). Many human disease genes that are required for normal cilia formation and function have C. elegans counterparts, including the ADPKD genes lov-1 and pkd-2. C. elegans LOV-1 (PC1 family) and PKD-2 (PC2 family) colocalize in cilia of male-specific sensory neurons and act in a sensory capacity (Barr and Sternberg, 1999Go; Barr et al., 2001Go). lov-1 and pkd-2 mutant males are defective in two sensory behaviors: response to contact with a potential mate and location of the mate's vulva (the Lov phenotype). Hence, C. elegans male mating behavior provides a powerful read-out for polycystin function and ciliary localization mechanisms.

In a previous study (Hu et al., 2006Go), we proposed that cilia use a down-regulation process to adjust polycystin ciliary localization and signaling. In C. elegans, hyper-phosphorylation of PKD-2 promotes removal of this channel from cilia, which may be due to the overactivated channel activity (Hu et al., 2006Go). In yeast, Drosophila, and mammalian cell culture, the STAM (signal-transducing adaptor molecule) and Hrs (hepatocyte growth factor regulated tyrosine kinase substrate) complex binds and sorts internalized and monoubiquitinated membrane proteins on the early endosome to the multivesicular body (MVB) for lysosomal degradation (Asao et al., 1997Go; Komada et al., 1997Go; Bilodeau et al., 2002Go; Lloyd et al., 2002Go; Raiborg et al., 2002Go; Shih et al., 2002Go; Takata et al., 2000Go; Bache et al., 2003Go; Mizuno et al., 2003Go). Whether sensory cells use this lysosomal targeting mechanism to down-regulate ciliary receptor signaling is not known.

Herein, we demonstrate that STAM physically associates with the LOV-1 C-terminus and the STAM-Hrs complex directs internalized LOV-1 and PKD-2 on endosomes at the ciliary base to lysosomal degradation pathway. These data provide insight into a mechanism that modulates polycystin ciliary abundance and sensory signaling and demonstrate that sensory cells use receptor down-regulation and degradation to fine-tune ciliary sensory transduction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Alleles
Nematode culturing and genetics were performed as described (Brenner, 1974Go). him-5(e1490) was used as the wild type (Hodgkin, 1983Go). stam-1 (ok406) was obtained from the Caenorhabditis Genetics Center (CGC). We constructed transgenic lines by injecting plasmid DNA (total 120 ng/µl) using standard protocols (Mello and Fire, 1995Go). In all experiments we used plasmid pBX1 containing the wild-type pha-1(+) gene as a cotransformation marker in the pha-1(ts) strain (Granato et al., 1994Go). STAM-1 and HGRS-1 plasmids were injected at 0.6 ng/µl, 6 ng/µl, and 60 ng/µl.

Molecular Biology Techniques
The full-length LOV-1::GFP plasmid was constructed by inserting GFP into the Eco47III site of a 13-kb genomic lov-1 fragment (Barr and Sternberg, 1999Go). LOV-1::GFP is able to restore lov-1(sy582) response and vulva location efficiencies from ~20% to 55–60% (n = 72). PKD-2::GFP is described in (Bae et al., 2006Go; Hu et al., 2006Go). All other expression plasmids were built by cloning gene-specific promoters and cDNAs into green fluorescent protein (GFP)-tagging Fire vector pPD95.75. By using cDNAs with or without a stop codon, we generated STAM-1A and HGRS-1 GFP-tagged or untagged constructs, respectively. For the pkd-2 promoter, a 1.3-kb genomic fragment upstream of the start codon was used (Hu and Barr, 2005Go). For the stam-1 promoter, a 1.5-kb genomic fragment upstream of the stam-1 start codon was used. For the Ppkd-2::Ubi-PKD-2::GFP construct, PCR was used to fuse a ubiquitin cDNA to the 5' end of the pkd-2 cDNA, followed by cloning into the Fire vector. Ppkd-2::STAM-1A{Delta}UIM was PCR-generated, removing amino acids 149-196.

Yeast Two-Hybrid Experiments
The yeast strain AH 109 (Clontech, Palo Alto, CA) was used for yeast two-hybrid (Y2H) experiments. A cDNA library derived from mixed-stage him-5 animals was constructed in the GAL4 activation domain (DNA-AD) vector, pGADGH (Hu and Barr, 2005Go). Bait proteins were expressed in the GAL4 DNA-binding domain (DNA-BD) vector, pGBKT7. The C-terminus of C. elegans LOV-1 (amino acids: 3056-3178) was used as bait in Y2H experiments. Different stam-1 fragments (see Figure 1) were cloned into the pGADT7 vector. Protein-protein interactions were accessed by growth rate on SD–Leu-Trp-His-Ade high stringency plates and beta-galactosidase filter assays.

Mating Behavior Assay
Mating behavior assays are scored as described (Barr and Sternberg, 1999Go). Response efficiency reflects the percentage of males successfully responding to hermaphrodite contact within 5 min. An individual male's vulva-location ability was calculated as the number of positive vulva locations divided by the total number of vulva encounters. Vulva-location efficiency indicates the average behavior of a genotypic population. In all experiments, at least 24 animals were scored per experimental trial. Triplicate trials were performed for each line to obtain statistical data. All behavioral assays were done with the experimenter completely blinded to the sample.

Imaging Analysis and Fluorescence Intensity Measurements
Epi-fluorescence microscopy experiments were carried out using a Zeiss Axioplan2 Imaging system (Thornwood, NY), and photographed with an Orca-ER camera. Confocal experiments were carried out on a Bio-Rad MRC-1024 laser scanning confocal microscope (Richmond, CA). To measure cilium/cell body fluorescence intensity ratio (Hu et al., 2006Go), L4 worms were picked and cultured at room temperature for 16–20 h. Confocal or epi-fluorescence images were taken on adults. Overexposure was avoided by making sure fluorescence was not saturated in any area on confocal images. Mean fluorescence intensities of the cilium region (including cilium proper, transition zone, and ciliary base), the corresponding cell body (including nucleus) and background (area without worm) were measured using Openlab software (Improvision, Lexington, MA). Cilium/cell body fluorescence intensity ratio was calculated by the following formula: (mean cilium fluorescence intensity – background fluorescence intensity)/(mean cell body fluorescence intensity – background fluorescence intensity).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
C. elegans STAM Interacts with the LOV-1 Carboxy Terminus
To identify proteins that associate with LOV-1 and participate in polycystin ciliary localization and/or signaling, we performed a yeast two-hybrid screen with the 128-amino acid LOV-1 cytoplasmic C-tail as bait. Of 1 x 106 cDNAs screened, the interactor STAM-1A was isolated twice. STAM-1A does not bind the C-tail of PKD-2, demonstrating a specific association with the LOV-1 C-tail.

stam-1 (also called pqn-19) encodes the single STAM homolog in the C. elegans genome. Originally named for a role in cytokine signaling, STAM (signal transducing adaptor molecule) forms a complex with Hrs (hepatocyte growth factor regulated tyrosine kinase substrate) on the cytoplasmic face of the early endosome. The STAM-Hrs complex sorts monoubiquitinated membrane receptors to MVBs for lysosomal degradation in yeast, Drosophila, and mammals. The C. elegans genome encodes one Hrs homolog, which has been referred to as hgrs-1 (hepatocyte growth factor-regulated tyrosine kinase substrate), pqn-9 (www.wormbase.org), and Cevps-27 (Roudier et al., 2005Go).

STAM protein family members share a similar domain organization (Figure 1A). To identify the LOV-1 binding site of STAM-1A, we examined the ability of each domain to associate with the LOV-1 C-tail. As shown in Figure 1B, the C-terminal proline (P)-glutamine (Q)-rich region (amino acids 302-457) interacts with the LOV-1 C-terminus. In contrast, N-terminal fragments containing the VPS-27/Hrs/STAM (VHS), ubiquitin-interacting motif (UIM), and Src homology 3 (SH3) domains (amino acids 1-301) together or separately exhibit no binding. These results indicate that the STAM-1A C-terminus is necessary and sufficient for LOV-1 binding. To narrow down the region of LOV-1 that interacts with STAM-1A, we split the LOV-1 C-terminus into two fragments corresponding to amino acids 3051-3119 and 3120-3178. Both fragments interact with STAM-1A in the Y2H assay, although the former was stronger (data not shown). These data indicate that STAM-1A may interact with the LOV-1 C-terminus at multiple sites.


Figure 1
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  		Figure 1. STAM-1A interacts with the LOV-1 C-terminus. (A) C. elegans STAM-1 has two splicing isoforms, STAM-1A and STAM-1B. STAM has a shortened PQ-rich domain plus a unique 9-amino acid stretch its C-terminus (represented by a red triangle). All STAM family members share a similar structure with a VHS (VPS-27/Hrs/STAM) domain, UIM (ubiquitin-interacting motif) domain, SH3 (Src homology 3) domain, and a proline/glutamine (PQ)-rich C-terminus. The C-terminus of vertebrate STAM contains an ITAM (immunoreceptor tyrosine-based activation motif) that binds SH2-domain–containing proteins. The ITAM domain is lacking in yeast, C. elegans, and Drosophila STAM proteins. The stam-1 genomic structure and ok406 deletion were reproduced from Wormbase (www.wormbase.org). (B) Y2H assays define the minimal region responsible for the interaction between STAM-1 and the LOV-1 C-terminus as the 155-aa STAM-1A C-terminus. The STAM-1B C-terminus does not interact with the LOV-1 C-terminus.

 
stam-1 has two splicing isoforms, stam-1a and stam-1b. STAM-1B (also called PQN-19B) has a shorter C-terminus (lacking the last 59 amino acids of STAM-1A) plus a 9-amino acid extension (Figure 1A). Interestingly, the STAM-1B C-terminus (amino acids 302-397) exhibited no binding (Figure 1B), indicating that the interaction with LOV-1 is STAM-1A isoform-specific.

STAM-1A Localizes to Endosomal-like Structures in Cell Bodies, Dendrites, and Ciliary Bases in Polycystin-expressing Male-specific Sensory Neurons
stam-1 is expressed in many tissues, including the pharyngeal intestinal valve, several head neurons, and phasmids in both males and hermaphrodites throughout development (data not shown). In males, stam-1 expression is also observed in the gonad and sensory neurons in the tail. stam-1 and pkd-2 are clearly coexpressed in male-specific ciliated CEM, ray B-type (RnB), and hook HOB sensory neurons (Figure 2B).


Figure 2
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  		Figure 2. STAM-1A coexpresses with LOV-1 and PKD-2 in male-specific neurons and shows an endosome-like distribution pattern. (A) Anatomy of C. elegans male and PKD-2–expressing neurons. Modified from (Hu et al., 2006Go). Top, differential interference contrast (DIC) side view image of an adult C. elegans male oriented head (left) to ventral up tail (right). Middle, lov-1 and pkd-2 are expressed in male head CEM neurons (left) and tail ray B type neurons (except R6B) and the hook HOB neuron (right). Bottom, confocal micrographs of PKD-2::GFP expression pattern in male head and tail. The cilium, dendrite, cell body, and axon of the CEMD (dorsal CEM) and CEMV (ventral CEM) neuron are drawn in the male head diagram. CEM cilia are in the nose region (dashed rectangular box). The pharynx is green. Positions of nuclei of all lov-1– and pkd-2–expressing cells in the C. elegans adult male tail (modified from Sulston et al., 1980Go). Ray neurons are arranged as nine left-right bilateral pairs, numbered 1–9, anterior to posterior (ventral up view shown here). Rays are required for response to contact with a potential mate (Liu and Sternberg, 1995Go). HOB is an asymmetric ciliated hook neuron that mediates vulva location behavior (Liu and Sternberg, 1995Go). For simplicity, only the dendrite of R3B left and HOB is shown. The R3B cilium is indicated by a dashed rectangular box. (B) Confocal micrographs of Pstam-1::DsRed2 (using the stam-1 promoter to drive DsRed2 expression) and PKD-2::GFP double-labeled C. elegans males. In all figures, adult males are oriented anterior to posterior, with the head pointing left and the tail pointing right. stam-1 coexpresses with pkd-2 in CEMs (head), RnBs, and HOB (tail, arrow points to HOB neuron). (C) With its native promoter, STAM-1A::GFP displays an endosomal-like pattern throughout the neuronal cell bodies. Note that STAM-1A also localizes to the cilia base of cilia in enlarged figure (right panel). In this and all subsequent figures, the arrow marks the ciliary base, and the arrowhead marks the approximate end of the cilium. (D) To clearly visualize the localization pattern of STAM-1A in polycystin-expressing neurons, the pkd-2 promoter is used to drive stam-1a cDNA expression only in CEMs, RnBs, and HOB neurons. STAM-1A localizes to the ciliary base (arrow), but is excluded from the cilium proper (region between arrow and arrowhead). Large green puncta are observed along distal dendrites of RnB ray neurons. Enlarged figures show the cilia region of CEM and R3B. Scale bars in rightmost C and D panels, 5 µm; in all other micrographs scale bars, 10 µm.

 
A GFP-tagged STAM-1A fusion protein (Pstam-1a::STAM- 1A::GFP, P stands for promoter) localized to cytoplasmic and dendritic puncta resembling endosomes, and appeared to accumulate at the ciliary base, which corresponds to the distal-most end of the dendrite and/or transition zone, but not the cilium proper (Figure 2C, enlarged panel). To more carefully examine STAM-1A subcellular distribution pattern, the pkd-2 promoter was used to restrict STAM-1A::GFP expression (Ppkd-2::STAM-1A::GFP) to male-specific sensory neurons. STAM-1A::GFP labels puncta and accumulates at the ciliary bases, but is obviously excluded from the cilium proper of CEM, RnB and HOB neurons (Figure 2D). In contrast to STAM-1A, both PKD-2 and LOV-1 localize to the ciliary base and cilium proper (Figure 2A). The collocation of STAM-1A, LOV-1, and PKD-2 at the ciliary base suggests that STAM-1A may play a role in polycystin trafficking.

STAM-1A and HGRS-1 Collocate with RAB-5 on Early Endosomes at the Ciliary Base
To confirm that STAM and Hrs also localize to early endosomes in C. elegans sensory neurons, we performed colocalization experiments with STAM, Hrs, and the early endosomal marker RAB-5 (Sato et al., 2005Go). STAM-1 and HGRS-1 expression completely overlaps in cell bodies and ciliary bases of polycystin-expressing neurons (Ppkd-2::STAM-1A:: DsRed2 and Ppkd-2::HGRS-1::GFP, Figure 3A). Moreover, STAM-1 and RAB-5 collocate in the cell bodies and ciliary bases of polycystin-expressing neurons (Ppkd-2::STAM-1A:: GFP and Ppkd-2::mRFP::RAB-5, Figure 3, B–D). Combined, these data indicate that C. elegans STAM-1A colocalizes with HGRS-1 on the early endosome at the ciliary base, where they may participate in endocytic sorting of LOV-1 and PKD-2.


Figure 3
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  		Figure 3. STAM-1A is an early endosomal protein that localizes with RAB-5 and HGRS-1 in male-specific neurons. (A) Confocal micrographs of Ppkd-2::STAM-1A:: DsRed2 and Ppkd-2::HGRS-1::GFP double-labeled males. STAM-1A and HGRS-1 colocalize in cell bodies, the bases of cilia, and along RnB dendrites. Like STAM-1, HGRS-1 does not localize to the cilium proper. Arrows point to colocalization of STAM-1A and HGRS-1 at the ciliary base of CEM and R3B. (B) Confocal micrographs of Ppkd-2::STAM-1A::GFP and Ppkd-2::mRFP::RAB-5 double-labeled males. RAB-5 is an early endosomal marker (Sato et al., 2005Go). STAM-1A colocalizes with RAB-5 in the cell body and ciliary base. (C) Higher magnification images show that RAB-5 and STAM-1A collocate in CEM cell body. (D) Higher magnification fluorescence and DIC/fluorescence images of CEM cilia. RAB-5 and STAM-1A are never observed in the cilium proper. Scale bars, (A and B) 10 µm, and (C and D) 5 µm.

 
STAM-1 Regulates Endosomal Sorting of LOV-1 and PKD-2
If STAM-1A and HGRS-1 are important for LOV-1 and PKD-2 endocytic sorting, then mutations that disrupt stam-1 or hrgs-1 should affect polycystin localization and/or function. hgrs-1 null mutations are larval lethal (Roudier et al., 2005Go), precluding analysis of adult phenotypes. RNA interference (RNAi) targeting stam-1a or stam-1b did not result in embryonic or larval lethality, suggesting that stam-1 is not essential for viability (Fraser et al., 2000Go; Sonnichsen et al., 2005Go). To genetically deduce the function of stam-1, we analyzed the stam-1(ok406) putative null allele that contains a large deletion from the third to final exons (Figure 1A). The ok406 allele deletes the majority of the STAM-1 protein (both A and B isoforms), including a portion of the VHS domain, the entire UIM and SH3 domains, and the majority of the PQ-rich C-terminus (Figure 1A). stam-1(ok406) hermaphrodites produce greater than 80% unfertilized and dead eggs. Consistent with this sterility defect, stam-1 was identified as a germline regulator of oocyte meiotic maturation in an RNAi screen (Govindan et al., 2006Go). Transgene silencing is common in the germline and would explain why we did not detect STAM-1::GFP in sperm or oocytes. These data suggest that stam-1 may have multiple sites of cellular action that include the germline and neurons.

The remaining viable stam-1(ok406) adult hermaphrodites exhibit normal locomotion, feeding, food sensation, and egg laying behaviors. Sensory cilia of stam-1 mutants are intact as judged by fluorescent dye filling of amphid and phasmid neurons (data not shown). stam-1(ok406) adult males exhibit a slight, but statistically significant defect in the response step of mating behavior (response efficiency: 79.3 ± 4.6% for stam-1(ok406) males versus 89.1 ± 2.1% for wild-type males, p < 0.05). The response defect is consistent with stam-1 regulating the polycystins.

In mice, knockout of STAM causes decreased levels of Hrs (Kanazawa et al., 2003Go). To determine if Hrs levels are similarly affected by disruption of stam-1 in C. elegans, we performed RT-PCR using mRNA isolated from stam-1 and wild-type animals. We find that hgrs-1 levels are normal in the stam-1 mutant background (data not shown).

To determine whether stam-1 regulates polycystin trafficking, we compared the localization of functional LOV-1::GFP and PKD-2::GFP fusion proteins in wild-type and stam-1 mutant males. lov-1 encodes a predicted 3178-amino acid protein, making cDNA structure–function–localization studies prohibitively difficult. Hence, a full-length LOV-1::GFP reporter was generated by inserting GFP into a rescuing lov-1 genomic clone between amino acids 3119 and 3220 (Barr and Sternberg 1999Go). LOV-1::GFP and PKD-2::GFP are able to rescue the response and Lov defects of the lov-1(sy582) and pkd-2(sy606) single mutants, respectively (Materials and Methods, Bae et al., 2006Go). Faint LOV-1::GFP expression is detected in CEM cilia (Figure 4A, left), but only rarely observed in hook HOB and ray RnB cilia (data not shown), which may be due to mosaicism or very low lov-1 expression levels. PKD-2::GFP is more strongly expressed than LOV-1::GFP, facilitating analysis of STAM-1 function in polycystin ciliary localization. In wild-type animals, PKD-2::GFP primarily localizes to the cell body, ciliary base, and cilium proper (Figure 4, C and D) and is observed moving bidirectionally along the dendrite (Bae et al., 2006Go).


Figure 4
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  		Figure 4. LOV-1 and PKD-2 accumulate at the ciliary base in the stam-1(ok406) mutant. Confocal micrographs of LOV-1::GFP and PKD-2::GFP transgenic males. (A) LOV-1::GFP localizes to the CEM cilium proper in wild type (left). In the stam-1 mutant, LOV-1::GFP and small puncta (hollow arrows) accumulate at the CEM ciliary base. Small vesicle-like puncta accumulate around the CEM ciliary base (hollow arrows). (B) Fluorescence intensity ratios measure increased ciliary base accumulation of LOV-1::GFP in the stam-1 mutant. (C) PKD-2::GFP accumulates at the CEM ciliary base in a stam-1 mutant. Small PKD-2::GFP puncta are observed below the CEM ciliary base (hollow arrows). Right panels show a higher magnification of the CEMs in the male nose. Top panels reproduced from Hu et al. (2006)Go. (D) In RnB neurons, PKD-2::GFP accumulates at the ciliary base and along the distal dendrites of stam-1 males (bottom) compared with wild type (top). High magnifications show R3B of wild-type (top) and stam-1 (bottom) males. In all figures, the arrowhead points to the tip of the cilium. The solid arrow points to the ciliary base. Hollow arrows point the puncta observed in the RnB distal dendrite. Scale bars in rightmost C and D, 5 µm; in all other micrographs scale bars, 10 µm. (E) Fluorescence intensity ratios measure increased ciliary base accumulation of PKD-2::GFP in the stam-1 mutant.

 
In stam-1(ok406) males, LOV-1::GFP and PKD-2::GFP localize normally to the cilium proper of CEM neurons (Figure 4, A and C). However, both LOV-1::GFP and PKD-2::GFP accumulate at the ciliary base (arrowhead in Figure 4, A and C) and are often observed in discrete puncta in the ciliary base region (thin arrows in Figure 4, A and C). In RnB neurons of stam-1 mutant males, many PKD-2::GFP–labeled puncta accumulate along the distal dendrite (Figure 4D, bottom). PKD-2 dendritic motility is intact, indicating that stam-1 is not required for transportation between the neuronal cell body and ciliary base. LOV-1 and PKD-2 subcellular distribution at ciliary base in stam-1(ok406) animals is similar to the STAM-1A::GFP localization pattern in wild-type male sensory neurons (compare Figure 4, A, C, and D, with Figure 2D), indicating that the polycystins may accumulate in endosomes in the absence of STAM. The finding that LOV-1 and PKD-2 accumulate at ciliary bases in a stam-1 mutant background is consistent with the evolutionarily conserved role for STAM in sorting membrane receptors for lysosomal degradation.

Accelerating Endosomal Sorting Affects PKD-2 Ciliary Function and Localization
If STAM-1A and HGRS-1 function to sort endocytosed LOV-1 and PKD-2 to the MVBs for lysosomal degradation, then accelerating this process may affect polycystin ciliary abundance and function. To test this hypothesis, we overexpressed each protein in male-specific sensory neurons and scored effects on male mating behavior and polycystin localization. We observed greater behavioral and targeting effects with increasing concentrations of injected Ppkd- 2::STAM-1A::GFP or Ppkd-2::HGRS-1::GFP DNA (Figure 5).


Figure 5
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  		Figure 5. Overexpression of STAM-1A or HGRS-1 interferes with polycystin mediated male sensory behaviors and polycystin ciliary localization. STAM-1A (A) or HGRS-1 (B) are injected into wild-type animals. Greater behavioral and targeting effects are observed with increasing concentrations of injected Ppkd-2::STAM-1A::GFP or Ppkd-2::HGRS-1::GFP DNA (0.6, 6, and 60 ng/µl). myEx stands for extrachromosomal array in transgenic animals. (C) To rule out potential effects of a GFP tag on STAM-1A and HGRS-1 in A and B, tagless Ppkd-2::STAM-1A and Ppkd-2::HGRS-1 constructs were injected at 60 ng/µl into wild-type animals. Similar defects in response and vulva location behaviors are observed with tagged and untagged versions of STAM-1A and HGRS-1. (D) Transgenic animals from C were crossed with another transgenic line containing an integrated, functional PKD-2::GFP to generate the strains PKD-2::GFP; myEx(Ppkd-2::STAM-1A) and PKD-2::GFP; myEx(Ppkd-2::HGRS-1). Fluorescence intensity ratios show that overexpression of STAM-1A or HGRS-1 dramatically reduces PKD-2 ciliary localization. (E) As negative controls, Ppkd-2::STAM-1A{Delta}UIM and Ppkd-2::STAM-1B constructs were injected at 60 ng/µl into wild-type animals. No response or location of vulva (Lov) defect is observed. (F) Transgenic animals obtained from E were crossed with PKD-2::GFP. Fluorescence intensity ratios show no change in PKD-2::GFP distribution with STAM-1A{Delta}UIM or STAM-1B overexpression. Mating behavior assays are described in Materials and Methods. Figures were generated using Sigmaplot5 (Jandel Scientific, San Rafael, CA). Data are represented as mean ± SEM. **p < 0.01 compared with control.

 
STAM-1A or HGRS-1 overexpression resulted in response and location of vulva (Lov) defects, similar to that of polycystin mutant males (Figure 5, A and B). Overexpression of GFP-tagged or untagged STAM-1A or HGRS-1 resulted in consistent and comparable response and Lov defects (Figure 5C). STAM-1A or HGRS-1 overexpression dramatically reduces the amount of PKD-2 in the cilium and ciliary base (Figure 5D). As a negative control, the STAM-1B isoform that does not interact with LOV-1 was overexpressed and has no effect on male mating behavior (Figure 5E) or PKD-2::GFP ciliary localization patterns (Figure 5F). This data indicates STAM-1A, but not STAM-1B, specifically acts in the polycystin down-regulation process. We propose that endocytosed PKD-2 may either be recycled or degraded and that overexpression of STAM-1A or HGRS-1 accelerates the latter process, thereby reducing PKD-2 ciliary signaling and membrane distribution.

STAM-1A Sorts Phosphorylated and Ubiquitinated PKD-2 for Lysosomal Degradation
The UIM domain of STAM and Hrs binds the ubiquitin moiety of ubiquitinated membrane receptors is critical for sorting and down-regulation (Bilodeau et al., 2002Go; Polo et al., 2002Go; Shih et al., 2002Go; Bache et al., 2003Go; Fisher et al., 2003Go; Hicke and Dunn, 2003Go; Mizuno et al., 2003Go; Swanson et al., 2003Go; Urbe et al., 2003Go; Komada and Kitamura, 2005Go). The UIM itself is required for monoubiquitination of sorting machinery proteins (such as Epsin, Eps15, Hrs, and STAM), suggesting that the UIM may function other than as a sorting determinant (Oldham et al., 2002Go; Polo et al., 2002Go). We generated a STAM-1A{Delta}UIM truncation protein lacking the UIM domain. Overexpression of STAM-1A{Delta}UIM does not affect male mating behaviors or PKD-2 ciliary localization (Figure 5, E and F) nor does STAM-1A{Delta}UIM rescue the PKD-2::GFP localization defects of a stam-1 mutant (data not shown), demonstrating an essential role for the UIM domain in polycystin down-regulation.

To test the hypothesis that ubiquitination plays a role in polycystin down-regulation, we directly coupled a 76-amino acid ubiquitin tag to the amino terminus of PKD-2 (Ubi-PKD-2::GFP). A similar approach was used by Kaplan and colleagues to show that direct conjugation of ubiquitin to the C. elegans glutamate receptor GLR-1 promotes endocytic removal at synapses (Burbea et al., 2002Go). Ubi-PKD-2:: GFP exhibits a completely normal cell body localization pattern but is largely absent from the cilium and ciliary base (Figure 6A) and cannot rescue the response and Lov defects of a pkd-2 mutant (Figure 6B). Ubi-PKD-2::GFP dendritic transport and ciliary targeting are intact (data not shown), suggesting that ciliary Ubi-PKD-2::GFP is targeted for degradation.


Figure 6
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  		Figure 6. stam-1 is involved in down-regulating ubiquitinated or hyper-phosphorylated PKD-2 from sensory cilia. (A) Ubi-PKD-2::GFP is observed in neuronal cell bodies but virtually absent from cilia. (B) Ubi-PKD-2 cannot rescue the response and vulva location defects of pkd-2(sy606) mutants. (C) In the stam-1(ok406) mutant, Ubi-PKD-2 accumulates at the ciliary base, which is similar to the PKD-2::GFP distribution pattern in the stam-1(ok406) mutant. In RnB ray neurons, Ubi-PKD-2::GFP puncta are observed along the distal dendrites. Right panels show a magnified view of the cilia region of the CEMs and R3B. (D) Fluorescence intensity ratios show an increase of Ubi-PKD-2 at the ciliary base in stam-1(ok406) mutant compared with Ubi-PKD-2 in wild type. (E) In wild type, PKD-2S534D::GFP is observed in neuronal cell bodies, but mostly absent from cilia (reproduced from Hu et al., 2006Go). (F) In the stam-1(ok406) mutants, PKD-2S534D accumulates at the ciliary base, which is similar to the Ubi-PKD-2 localization pattern in stam-1(ok406) mutants. In RnB neurons, small PKD-2S534D::GFP puncta distribute along the distal dendrites. Right panels show a higher magnification of the cilia region of CEM and R3B neurons. (G) Fluorescence intensity ratios demonstrate an increase of PKD-2S534D at the ciliary base in stam-1(ok406) mutants when compared with PKD-2S534D in wild type. Data are represented as mean ± SEM. **p < 0.01 compared with control. Scale bars in rightmost C and F panels, 5 µm; in all other micrographs scale bars, 10 µm.

 
In general, ubiquitinated proteins may be degraded via the proteasome or lysosome. Efficient degradation of ubiquitinated proteins by the proteasome requires the formation of polyubiquitin chains containing at least four ubiquitin moieties at ubiquitin K48 site (Thrower et al., 2000Go). Conversely, monoubiquitination is sufficient for sorting into MVBs and subsequent lysosomal degradation, a mechanism mediated by the STAM-Hrs complex. We tagged PKD-2 with the ubiquitin mutant Ubi(K48R), which cannot be polyubiquitinated. Ubi(K48R)-PKD-2::GFP and Ubi-PKD-2::GFP localization patterns are identical (data not shown), suggesting that PKD-2 degradation does not require K48 polyubiquitination and is likely due to STAM-Hrs–mediated lysosomal degradation. Next, we reasoned that by blocking STAM-1, Ubi-PKD-2 should accumulate at the ciliary base. Consistent with this hypothesis, Ubi-PKD-2::GFP localizes to the ciliary bases of CEM and RnB neurons in the stam-1 mutant (Figure 6C). In the stam-1 mutant, fluorescence intensity values for Ubi-PKD-2::GFP at the ciliary base increased 10-fold compared with a wild-type background (Figure 6D). In RnB neurons, Ubi-PKD-2::GFP puncta are also observed in the distal dendrite. Unfortunately, attempts at detecting ubiquitinated PKD-2 in vivo have been unsuccessful, likely due to the difficulty of detecting endogenous PKD-2 on a Western blot. These data suggest that STAM-1A acts on early endosomes located at the ciliary base and sorts LOV-1 and PKD-2 for lysosomal degradation.

We next determined whether retrograde transport by the CHE-3 dynein or cytoplasmic dynein retrograde motor (Signor et al., 1999Go; Wicks et al., 2000Go; Koushika et al., 2004Go) is required for STAM-1–mediated polycystin down-regulation. We examined Ubi-PKD-2::GFP in che-3(e1379) and dhc-1(or195) (dynein heavy chain) single mutants. Similar to the wild-type background, Ubi-1::PKD-2::GFP levels are greatly reduced in che-3 and dhc-1 mutant males (data not shown). These observations suggest that che-3 and dhc-1 either act upstream of stam-1 or do not function at all in the PKD-2 down-regulation process.

To further explore the effects of posttranslational modifications on ciliary receptor down-regulation, we made use of the "phosphomimetic" PKD-2S534D mutant. Casein kinase 2 (CK2) and calcineurin (TAX-6) act in concert to regulate the phosphorylation state of PKD-2 at the S534 site (Hu et al., 2006Go). When S534 is constitutively phosphorylated via the S534D mutation, PKD-2 ciliary abundance, but not initial ciliary targeting, is dramatically reduced. We proposed that PKD-2S534D may represent an overactivated channel whose activity is signaling down-regulated via removal from cilia. To test this possibility, we examined PKD-2S534D::GFP localization in the stam-1(ok406) mutant. In stam-1 animals, PKDS534D::GFP localizes to the ciliary base, and the fluorescence intensity ratios for PKD-2S534D::GFP increase about fivefold (Figure 6F). Strikingly, wild-type PKD-2::GFP, Ubi-PKD-2::GFP, and PKD-2S534D::GFP accumulate at the stam-1(ok406) ciliary base in a similar pattern (compare Figure 3, C and D, with Figure 6, C and E, respectively). These data suggest that PKD-2S534D and Ubi-PKD-2 are down-regulated and degraded via the same mechanism. Cumulatively, our data indicate that STAM-1A binds, sorts, and targets the polycystin complex for lysosomal degradation (Figure 7).


Figure 7
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  		Figure 7. Model of the TRPP down-regulation and degradation process. In C. elegans sensory cilia, LOV-1 and PKD-2 might assemble into a receptor-channel complex as established for the mammalian polycystins in renal primary cilia (Nauli et al., 2003Go). Activation of polycystins causes an increase in intracellular Ca2+. Polycystin signaling and localization is controlled by a dynamic phosphorylation cycle (Cai et al., 2004Go; Kottgen et al., 2005Go; Hu et al., 2006Go). Here we show that LOV-1 and PKD-2 signaling and localization are also regulated by ubiquitination and internalization at the ciliary base. The LOV-1 C-tail serves as a recognition signal to recruit the STAM-1-HGRS-1 (STAM-Hrs) sorting machinery to the polycystin complex. The STAM-Hrs machinery then sorts the polycystin complex the lysosome for degradation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Receptor down-regulation protects an organism from uncontrolled signaling, cell growth, and proliferation (Katzmann et al., 2002Go; Staub and Rotin, 2006Go). For example, ligand-activated EGFRs on the plasma membrane are internalized and sorted for lysosomal degradation (Schlessinger, 2000Go). Our report is the first to show that ciliary receptors and TRP channels may be down-regulated in a similar manner. Here, we demonstrate that down-regulation of the TRP polycystin complex involves phosphorylation, ubiquitination, endocytic sorting, and lysosomal degradation. Whether a similar TRPP down-regulation mechanism acts in mammals is not known, although the HECT ubiquitin ligase AIP4 has been recently shown to regulate the cell surface expression of TRPV4 and TRPC4, but not TRPP2 (Wegierski et al., 2006Go). We propose that STAM-Hrs–mediated down-regulation controls ciliary TRPP receptor abundance and signaling. Too much or too little polycystin signaling results in a sensory defect in C. elegans males, and in mammals, may potentially result in PKD.

The STAM-Hrs complex acts on the early endosomes to sort internalized membrane proteins to MVB (Bilodeau et al., 2003Go; Mizuno et al., 2003Go; Raiborg et al., 2003Go). Our report provides evidence that a membrane receptor (LOV-1) is cargo of the STAM-Hrs complex. Surprisingly, the LOV-1-STAM-1 interaction does not require the UIM domain. How cargo proteins are recognized by STAM-Hrs complex is not well understood. One model involves binding of ubiquitinated cargo to the UIM domain found in STAM, Hrs, and many other components of the endocytic sorting machinery. Other lines of evidence hint at the existence of alternative recognition and sorting mechanisms (Raiborg et al., 2002Go) and suggest that the UIM may have functions other than as a sorting determinant. We propose that LOV-1 functions as an adaptor to recruit STAM-1 and Hrs in close proximity to ubiquitinated PKD-2. From here, the LOV-1-PKD-2 complex may be trafficked to the MVB. Without the LOV-1–interacting PQ-domain, STAM-1 is not recruited to the polycystin unit; without the UIM domain, STAM-1 cannot function to down-regulate PKD-2. We failed to detect an interaction between the STAM-1 UIM and the PKD-2 C-tail (data not shown), which indicates that 1) STAM-1 and PKD-2 do not directly interact, 2) an interaction requires PKD-2 to be ubiquitinated, or 3) the interaction is transient or occurs outside the PKD-2 region tested.

Many membrane proteins are down-regulated. The internalization of some membrane proteins, such as CXCR4, EGFR, and Ste3, is ubiquitination-independent (Levkowitz et al., 1998Go; Thien et al., 2001Go; Chen and Davis, 2002Go; Marchese et al., 2003Go), whereas other studies show that ubiquitination is essential for the rapid internalization of growth hormone receptor and Ste2 (Govers et al., 1999Go; Dunn and Hicke, 2001Go). After internalization, these receptors may traverse one of two routes: recycling back to membrane or sorting to the lysosome for degradation. Cell- or receptor-specific mechanisms may exist to ensure the specificity and efficiency of the down-regulation. Thus, other adaptor proteins may be involved in targeting endocytosed membrane proteins to the MVB. On the early endosome, STAM-1A may bind LOV-1 and recruit the sorting machinery to the polycystin complex. Potential interactions between UIMs of other sorting components and the ubiquitinated polycystins may ensure efficient sorting and degradation.

Our data suggest that a phosphorylated and/or ubiquitinated polycystin complex is down-regulated via the STAM-Hrs machinery (Figure 7). The subcellular localization point at which these post-translational modifications occur is not known. Overexpression of STAM-1A or HGRS-1 results in similar phenotypes: male response and Lov defects and a dramatic reduction of PKD-2 levels in the ciliary region. Conversely, knockout of stam-1 has the opposite effect on PKD-2 distribution: an accumulation at the ciliary base and distal dendrite. We interpret our results to mean that STAM-1 and HGRS-1 act to promote ciliary receptor down-regulation and that blocking this pathway results in a failure to degrade LOV-1 and PKD-2. A similar process is observed in Drosophila, where Hrs acts to promote degradation of activated receptors (Lloyd et al., 2002Go). In contrast, Hrs overexpression in mammalian cells inhibits lysosomal trafficking of ubiquitinated receptors (Chin et al., 2001Go; Petiot et al., 2003Go). The reasons for these experimental discrepancies are not known, but may reflect differences between in vivo and cultured cell systems.

Interestingly, ciliary and flagellar proteomes contain components of the ubiquitination machinery (Pazour et al., 2005Go; Liu et al., 2007Go). PC-1 interacts with the seven in Absentia (Siah-1) E3 ligase (Kim et al., 2004Go), but the physiological relevance of this association is not known. Although sharing no obvious sequence homology, the short C-terminal tails of both human PC-1 and C. elegans LOV-1 are essential for their respective roles in kidney epithelial cells and worm sensory neurons (Barr and Sternberg, 1999Go; Vandorpe et al., 2001Go; Nickel et al., 2002Go; Aguiari et al., 2003Go; Hooper et al., 2003Go; Chauvet et al., 2004Go; Kim et al., 2004Go). The C. elegans EGFR LET-23, GPCR ODR-10, and TRPP signal transduction pathways are negatively regulated by the AP-1 mu1 clathrin adaptor UNC-101 (Lee et al., 1994Go; Dwyer et al., 2001Go; Bae et al., 2006Go). Whether the STAM-Hrs complex acts in a common ciliary receptor down-regulation pathway remains to be seen.


   ACKNOWLEDGMENTS
 
We thank Dr. A. Fire (Stanford University) for plasmids and the Caenorhabditis Genetics Center for strains; D. Braun for excellent technical assistance; Dr. Barth D. Grant (Rutgers University) for the gift of mRFP::RAB-5 plasmid; and Dean Jeanette Roberts for intramural funding. This research is supported by grants from the PKD Foundation (J.H.) and National Institutes of Health (M.M.B.).


   Footnotes
 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-03-0239) on June 20, 2007.

{ddagger} Present address: Department of Genetics, Rutgers University, Piscataway, NJ 08854. Back

Address correspondence to: Maureen Barr (barr{at}biology.rutgers.edu).


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