the condition predicates heart disease and diabetes  two pathologies that diminish the quality of life and increase risk of premature death . while appetite control is incredibly complex , we do know that a hormone discovered in teleost fish , melanin - concentrating hormone ( mch ) , plays a role in regulating feeding behavior in higher - order mammals [ 15 ] . mch acts via a g protein - coupled receptor ( gpcr ) at the plasma membrane of many cell types including neurons to stimulate appetite and adipocytes to stimulate the synthesis and secretion of leptin . studies with mchr1 knockout mice have shown similar physiological adaptations including an increase in resistance to diet - induced obesity and hyperphagia compared to wild - type mice fed a similar diet . recently , loss of mchr1 localization to primary cilia has been linked to the severe obesity seen with bardet - biedl syndrome , suggesting that regulation of mch signaling plays an important role in energy homeostasis and that this pathway could become a pharmacological target to curb appetite . the usefulness of an mchr1 antagonist in treating human obesity is being explored by others ; an early phase i clinical trial has seen promising results . the dramatic phenotype observed in loss of function studies suggests that irregular mch signaling patterns have the potential to cause disease . the desensitization of mchr1 was first described by lembo et al . , who observed internalization of mchr1 by fluorescence microscopy following agonist treatment . this was supported by evidence that mchr1 internalization occurs via the canonical -arrestin and clathrin - mediated pathway [ 10 , 11 ] . flag - tagged mchr1 internalized in hek293 t cells following 30  min mch treatment to only 44.2% of initial surface receptor levels . further studies suggested that -arrestin 2 is preferentially recruited to mchr1 ; however , they were performed in the presence of overexpressed grk2 , a kinase that increases the affinity of gpcrs for arrestins . the goal of this study was to further characterize the agonist - mediated desensitization of mchr1 . we unexpectedly discovered early on that despite rapid and extensive desensitization of erk activation in response to mch , agonist - mediated internalization of mchr1 was very poor in our model , which is contrary to what others have reported [ 10 , 11 ] . although we were able to significantly improve receptor internalization by overexpression of -arrestins or grk2 , this study suggests that there is an unexplored desensitization mechanism that occurs at the plasma membrane in the absence of receptor internalization . bhk-570 cells ( atcc ) were cultured as a tissue monolayer using dmem media ( cellgro ) containing 10% fetal bovine serum ( atlanta biological ) without antibiotics . plasmid dna encoding mchr1 in pcdna3 was purchased from the missouri s&t cdna resource center . j. benovic at thomas jefferson university generously provided plasmids encoding -arrestin 1 and -arrestin 2 in pegfp - n as well as grk2 and grk2 k220r in prk5 . the leptin promoter - driven luciferase reporter plasmid p(-762)ob luc was a gift from dr . cell transfections were performed on cells plated for at least 24  h with lipod293 reagent following the recommended protocol from signagen . media were changed 5 hours after transfection and experiments were run approximately 48 hours after transfection . bhk-570 cells in 35  mm dishes were transfected with 1  g mchr1 in pcdna3 or empty plasmid on day 1 . on day 2 , cells were serum starved in dmem for 1824 hours . on the third day , baseline erk activation was obtained by treating cells for up to 30  min with 1  m mch ( rat , american peptide ) prior to harvesting in 2x laemmli sample buffer . desensitization assays were performed by treating cells with a desensitizing dose of mch for 15  min followed by rinsing with and incubation of cells in dmem in the incubator for 30  min ( called washout ) . to activate the pathway again , cells were treated with a second dose of mch for up to 30  min prior to harvesting in 2x laemmli sample buffer . lysates were boiled for 5  min and spun at top speed in a microcentrifuge prior to sds - page . proteins were transferred to nitrocellulose and western blots performed with antibodies to both phosphorylated and total erk ( cell signaling ) . bands were exposed using western lightning enhanced chemiluminescence kit ( perkin elmer ) on kodak film . coverslips seeded with bhk-570 cells were transfected with mchr1-vsvg  gfp - tagged -arrestin 1 or 2 . cells were then incubated for approximately 24  h and then treated with 1  m mch ( american peptide ) for up to 30  min . the coverslips were then rinsed twice with ice - cold pbs before fixing with 4% paraformaldehyde in pbs for 10  min at room temperature . after 3 more washes with pbs , they were transferred to a humidification chamber for incubation in blocking buffer ( pbs , 5% goat serum , 0.1% triton x-100 ) for at least 20  min . they were then incubated for at least 1  h with rabbit anti - vsvg polyclonal antibody ( sigma ) at 1  :  50 in blocking buffer . following 3 pbs washes , dapi ( roche ) was added at 1  :  500 and alexa fluor 546 goat anti - rabbit secondary antibody ( invitrogen ) at 1  :  250 in blocking buffer for 1  h. after 3 more pbs washes , coverslips were mounted on glass slides with prolong gold ( invitrogen ) . fluorescence micrographs were obtained with a zeiss axioskop fluorescence microscope with a zeiss axiocam camera and zeiss axiovision software . image cropping was performed in adobe photoshop and figures assembled in ms powerpoint . for figure 4 , twenty - four well plates were seeded with bhk-570 cells and transfected with mchr1-vsvg  gfp -arrestin 1 or 2 or in a separate experiment grk2 or k220r grk2 . twenty - four hours after transfection , the cells were incubated in labeling buffer ( dmem , 5% goat serum , 0.02  mm hepes ) with 1  :  1,000 mouse anti - vsvg ( sigma ) for 2  h. the cells were then washed twice in labeling buffer before being treated with 1  m mch for up to 30  min . the cells were then washed once with ice cold pbs and fixed with 3% paraformaldehyde in pbs for 20  min at room temperature . following 2 , 5  min washes with pbs , cells were incubated with goat anti - mouse hrp - conjugated secondary antibody ( bio - rad ) in pbs with 5% goat serum for 45 minutes . after another 3 pbs washes , 175  l of pod blue ( roche ) was added to develop for 3  min on an orbital shaker . one hundred fifty microliters from each well were transferred to a 96-well plate and the absorbance was read at 450  nm using a synergy 1 or elx800 plate reader ( biotek ) . bhk-570 cells were triple transfected with either ( 1 ) mchr1/p(-762)ob luc / gfp in pcdna3 , ( 2 ) mchr1/p(-762)ob luc / gfp--arrestin 1 , or ( 3 ) mchr1/p(-762)ob luc / gfp -arrestin 2 . the cells were treated 48  h after transfection with 1  m mch for up to 6  h in dmem and then lysed and luminol was added . light production was measured over 15  sec on a biotek synergy h1 plate reader . the averages of the data were reported  the standard error of the mean ( sem ) . student 's t - test was performed to determine statistical significance with data scoring equal to or greater than the 95th percentile considered significant . it was previously reported that mch caused activation of the mapk pathway and leptin promoter in 3t3-l1 adipocytes and that mchr1 was subsequently downregulated in these cells . to see if mchr1 could signal to erk in our model system , we transfected bhk-570 cells with mchr1 and stimulated them with mch for 0 , 2 , 10 , and 30  min prior to lysis . we observed transient erk activation in response to mch that was not detectable in empty vector transfected control cells as seen in figure 1(a ) as baseline activation , which is quantitated in figure 1(b ) by normalizing activated erk to total erk ( blots not shown ) . within 10  min we measured a greater than 30-fold activation of erk over baseline that disappeared by 30  min . surprisingly , when hormone was removed , cells were washed extensively and allowed to recover for up to 30  min and mch did not regain its ability to activate erk , indicating significant desensitization of the pathway ( figures 1(a ) and 1(b ) ) . we previously showed that an n - terminally tagged vsvg - mchr1 construct efficiently activates erk in response to mch binding , and preliminary desensitization experiments conducted with this construct in cho - k1 cells had similar results to those in figure 1 . therefore , we conclude that the presence of the vsvg tag does not interfere with receptor signaling ( data not shown ) , and we took advantage of the sensitivity of this epitope to explore the role that receptor internalization plays in controlling the duration of mch - mediated erk signaling . the desensitization of mchr1 was first described by lembo et al . , who observed internalization of mchr1 through fluorescence microscopy following agonist treatment . we hypothesized that internalization of mchr1 could be responsible for the extensive desensitization of the erk signaling pathway by mch . much to our surprise , we have been unable to observe any agonist - induced redistribution of mchr1 using vsvg - tagged mchr1 expressed in bhk-570 cells by fluorescence microscopy ( figure 2(a ) ) . this was neither specific to the construct ( mchr1-eyfp did not internalize ) nor was it specific to bhk-570 cells ( both cho - k1 cells and 3t3-l1 cells failed to internalize mchr1 ) ( data not shown ) . we thought that perhaps our method was not sensitive enough to capture the receptors being internalized so we decided to utilize a modified cell - surface elisa   to measure agonist - mediated internalization of an n - terminal vsvg - tagged mchr1 . the vsvg - tag allowed us to utilize a high - affinity antibody for this assay . we measured surface - localized receptor in untreated cells ( 100%  2.3% ) , and compared to cells treated with mch for 15 or 30  min , the results of which are shown in figure 2(b ) . it was quite surprising that mch was only able to drive surface receptor levels after 15  min to 96%  2.7% of control and after 30  min to 85%  2.5% ( p < 0.0025 ) of control , which was confirmed by fluorescence microscopy ( figure 2(a ) ) . to determine if high expression levels of vsvg - mchr1 contributed to the low internalization levels , we titrated back plasmid dna amounts during transfection and found that it did not improve internalization ( data not shown ) . this low level of internalization was also not a consequence of the n - terminal tag because murdoch and colleagues were able to observe internalization of this construct in hek 293 cells by fluorescence microscopy . others utilized flow cytometry to measure mch - mediated removal of mchr1 from the plasma membrane [ 10 , 11 ] . we wondered whether mchr1 could interact with the machinery to internalize in our model system , and so we decided to coexpress vsvg - mchr1 with either -arrestin 1 or -arrestin 2 and again measure internalization . overexpression of -arrestins has been shown to increase the internalization of 2-adrenergic receptors   and it was hypothesized that -arrestin would increase the rate of mchr1 internalization in response to mch . when -arrestin 1 was overexpressed , surface - localized mchr1 unexpectedly declined in the absence of agonist from 100%  3.4% to 92%  2.6% at 15  min and 79%  2.2% ( p < 0.01 ) at 30  min . treatment with mch however further improved the removal of mchr1 from the cell surface to 88%  2.9% of the total at 15  min and only 64%  2.4% after 30 minutes ( figure 3(a ) ) . when this experiment was repeated with -arrestin 2 , in contrast to -arrestin 1 we found steady surface levels of mchr1 over the course of 30  min in the absence of agonist . when cells were treated with hormone for 15  min , surface receptor levels fell from 100%  6.5% to 83%  4.6% and by 30  min declined to 62%  4.0% ( p we directly compared the effects that each -arrestin has on loss of mchr1 from the cell surface of bhk-570 cells treated with mch for 30  min in figure 4 . in our most successful internalization experiments , 60% of the receptor still remained on the plasma membrane despite vast overexpression of arrestins ( data not shown ) and supraphysiological agonist concentrations . coexpression of mchr1 with -arrestin 1 resulted in an agonist - independent net loss of 17.0% of mchr1 from the plasma membrane and an agonist - dependent net loss of 21.1% . the coexpression of -arrestin 2 resulted in no appreciable agonist - independent net loss and an agonist - dependent net loss of 24.7% ( p these experiments highlight some differences between the association amongst the receptor and each arrestin ; while both arrestins facilitated coupling of mchr1 with the internalization machinery , -arrestin 1 seems to have facilitated receptor coupling in the absence of agonist . to verify this visually -arrestin 1 overexpression seems to result in a large amount of both receptor and arrestin to be stuck in a juxtanuclear compartment ( figure 4(b ) ) . this could either be the result of inefficient trafficking of newly synthesized receptor to the plasma membrane or excessive removal of unoccupied receptors from the plasma membrane . although some -arrestin 1 was not visibly recruited to the plasma membrane in these cells ( figure 4(b ) , middle panel ) , treatment of cells with mch for 30  min did show mchr1 accumulation in punctate vesicles ( figure 4(b ) , top panel ) ; these vesicles lacked -arrestin 1 ( figure 4(b ) , bottom panel ) . -arrestin 2 gave a similar , but more extensive internalization profile with a larger number of mchr1-positive puncta ( figure 4(c ) , top panel ) that are also positive for gfp--arrestin 2 when the overlay is examined ( figure 4(c ) middle and bottom panels ) . this data agrees with evans and colleagues by demonstrating that the mchr1--arrestin 2 interaction is of higher affinity for the receptor than that of -arrestin 1 . to further explore the potential colocalization of vsvg - mchr1 with both -arrestins , shown in figure 5 is a representative single plane where colocalization of neither -arrestin with vsvg - mchr1 is readily apparent after a 30  min hormone treatment . instead , most mch receptor - containing vesicles are distinctly separate from -arrestin - associated vesicles . g protein - coupled receptor kinase 2 ( grk2 ) is a family member of a group of protein kinases that generally function to regulate gpcr signaling through the phosphorylation of serine and threonine residues located on the intracellular loops and c - terminal tails of gpcrs . the addition of phosphate groups to these areas has been shown to terminate signaling by inhibiting g protein coupling and facilitating receptor internalization by increasing the affinity of arrestins for the receptor . grk2 plays a role in both -arrestin - dependent and -arrestin - independent trafficking of gpcrs . a dominant - negative form of grk2 , grk2 k220r has already been characterized   and is known to knock down the expression of wild - type grk2 when coexpressed . based on previous studies , it was hypothesized that the overexpression of grk2 would increase the rate of internalization , while transfection of the k220r grk2 would knock down endogenous grk2 resulting in inhibited mchr1 internalization . mchr1 internalization was monitored by cell - surface elisa over the course of 30  min . cotransfection of mchr1 with either grk2 or k220r grk2 resulted in no appreciable loss of receptors in the absence of agonist ( figure 6 ) . < 0.005 ) for grk2-expressing cells was measured and a net increase in surface receptor levels of 11.3% ( p < 0.005 ) for k220r - expressing cells was measured when compared to cells given a control plasmid . in order to determine if this loss of receptor from the cell surface translates into improved desensitization of mch signaling , we utilized a reporter plasmid from which luciferase expression is driven by activation of the leptin promoter . this construct was previously reported to respond to mch over the course of several hours . as shown in figure 7 , cells expressing -arrestin 1 or 2 showed less luciferase expression compared to control in response to mch treatment for up to 6  h. this indicates that mch - mediated activation of the leptin promoter can be desensitized by overexpression of arrestins and it supports the hypothesis that agonist - mediated internalization of mchr1 acts to desensitize cells to mch . melanin - concentrating hormone acts on at least two g protein - coupled receptors in the central nervous system and peripheral organs to elucidate a variety of physiological responses . a few early studies suggested that at least one of those receptors , melanin - concentrating hormone receptor 1 , undergoes phosphorylation , -arrestin 2 recruitment , and agonist - mediated internalization [ 911 ] . however , when we attempted to replicate these experiments much to our surprise we were unable to measure internalization of mchr1 visually ( figure 2(a ) ) and when measured quantitatively , only weak ( ~15% ) internalization was observed ( figures 2(b ) and 3 ) despite extensive desensitization of the erk pathway ( figure 1 ) . our observations were not limited to bhk-570 cells , but mchr1 internalization was also not observed in cho - k1 cells or 3t3-l1 cells for various receptor constructs including mchr1-eyfp ( data not shown ) . this is in contrast to results described by others who observed internalization of mchr1 by other means [ 911 ] . the clathrin - mediated endocytosis pathway has been widely characterized from studies of other gpcrs . -arrestins are important mediators , helping to recruit clathrin heavy chain to gpcrs , and receptors such as the 2-adrenergic receptor are known to desensitize through this pathway [ 2123 ] . the relative levels of gpcr , -arrestin , and grk have been previously implicated in sequestration efficiency as reported by barak and colleagues for the 2-adrenergic receptor ; however , these differences probably do not entirely explain the results we see with mchr1 . mglu1r   and ghrh - r   are two gpcrs that when heterologously expressed in bhk-570 cells internalized rapidly following agonist exposure , suggesting that baby hamster kidney fibroblast cells indeed have the capacity to sequester gpcrs . when we coexpressed -arrestins 1 and 2 or grk2 with mchr1 , we were able to partially rescue internalization of the receptor ( figures 3 - 4 ) . our results suggest that -arrestin 1 is indeed capable of coupling mchr1 to the endocytic machinery , but that the association is weak because cointernalization of the arrestin with the receptor was not observed ( figure 4(b ) ) . we also presented evidence of agonist - independent removal of mchr1 from the cell surface with -arrestin 1 in figures 3(a ) and 4(a ) , although this could be the result of an antibody - induced conformational change that results in internalization of the receptor . when -arrestin 2 was coexpressed , co - internalization with mchr1 following agonist treatment was observed ( figure 4(c ) ) , but not confirmed with confocal microscopy methods ( figure 5 ) . these results mirror those from a study done with 2 adrenergic receptor in which cells triple transfected with 2-adrenergic receptor , 2-adrenergic receptor kinase , and -arrestins showed a significant synergistic effect . previously reported selective , but transient recruitment of -arrestin 2 to the plasma membrane of transfected hek293 cells following mch exposure . unlike their study , in our experimental system utilizing bhk-570 cells it was not necessary to overexpress grk2 to observe either -arrestins ' effect on mchr1 internalization . also , although we did not specifically detect recruitment of the gfp -arrestins to the cell surface , it is implied since their presence efficiently facilitated coalescing of receptors into punctate vesicles for internalization . saito and colleagues , using hek293 t cells , overexpressed -arrestin 2 and showed no significant effects on mch - mediated receptor internalization . these seemingly conflicting results may have been the result of inefficient grk2 levels to accompany the overexpressed -arrestin 2 . the best illustration of cell - to - cell variation in internalization efficiency is for the 2-adrenergic receptor , and abnormalities in grk or -arrestin protein levels have been linked to alzheimer 's disease , inflammatory diseases , and cardiac ailments . our results illustrate that protein levels of both grk2 and -arrestin have the potential to modulate the response of cells to mch , potentially influencing human appetite . g protein - coupled receptors can be divided into two classes : class a receptors and class b receptors . class a receptors preferentially bind -arrestin 2 over -arrestin 1 and their interaction is transient in nature with -arrestin and the receptor dissociating prior to entering the endosome . class b receptors have equal affinity for both -arrestin 1 and -arrestin 2 and form stable interactions resulting in the translocation of both receptor and -arrestin into the endosome . when both -arrestins were coexpressed with mchr1 in this study , receptor internalization was facilitated significantly over control , but to a greater extent with -arrestin 2 , suggesting that mchr1 is a class a receptor . if cointernalization of -arrestin 2 with mchr1 really does occur in our model system , it would suggest that mchr1 is a class b receptor . the protein kinase grk2 is known to phosphorylate serine threonine residues on the third intracellular loop and c - terminal tail of gpcrs increasing their affinity for -arrestins in an agonist - dependant manner . up until now , the effect of grk2 overexpression on the desensitization of mchr1 had yet to be explicitly measured although , as already stated , evans et al . utilized grk2 transfected cells while observing the nature of the interaction of -arrestins with mchr1 . agonist - induced internalization of mchr1 while overexpressing dominant - negative grk 2 showed no significant increase in the rate of receptor internalization ( figure 5 ) similar to the -opioid receptors . these receptors are dependent on phosphorylation by grks for the recruitment of -arrestins in contrast to that of the 2 adrenergic receptor , which can interact with -arrestins in the absence of grk2 phosphorylation sites [ 23 , 24 ] . this suggests that mchr1 is dependent on grk2 phosphorylation for the recruitment of -arrestins and together with previous data and that the rate - limiting step in mchr1 desensitization is dependent on the cellular concentration of -arrestins and/or grk2 . this would be true , however , if internalization was required for desensitization of mch signaling . yet erk signaling was efficiently desensitized ( figure 1 ) , we hypothesize that desensitization of the mch signaling pathway in these cells does not solely rely on removal of receptors from the plasma membrane . we hypothesized that overexpression of arrestins would not only promote agonist - mediated internalization of mchr1 , but that this would indeed translate into a desensitization of downstream signaling . our results in figure 7 support a role for -arrestins in downregulating this pathway because mch - mediated luciferase accumulation is essentially absent when they are coexpressed with mchr1 . this suggests that mch - mediated erk signaling is spatially linked to membrane - localized mchr1 and that agonist - mediated internalization of mchr1 is not necessary for erk signaling , rather it may participate in signal termination . mchr1 signaling to g proteins is thought to be regulated differentially by at least two rgs proteins , rgs2 and rgs8 [ 25 , 26 ] . mch - mediated erk signaling in hek293 cells proceeds via gq and gi , both of which are targets of rgs8 [ 25 , 26 ] . it is not known whether rgs8 plays a role in desensitizing erk signaling in bhk-570 cells , but it seems a likely candidate . another likely possibility is that mchr1 becomes spatially segregated from its signaling components in the plasma membrane . mchr1 forms a complex with caveolin-1 in these cells , and mchr1 is highly enriched in caveolae . it has been reported that gi and gs are able to migrate into and out of caveolae unlike gq , which is tethered there . interestingly , adipocytes express exceedingly high levels of caveolin-1 ; therefore regulation of mch signaling via caveolae or other lipid rafts in this cell type seems to be a strong possibility . the obese phenotype seen as a result of primary cilia loss in bardet - biedl syndrome is hypothesized to be the result of a loss of cilia - localized mchr1 in the brain . since mch signals appetite , this suggests that ciliary localization of mchr1 dampens the mch signal , further evidence to suggest that spatial organization of these receptors in the plasma membrane contributes to the regulation of its activity . we tested the hypothesis that agonist - induced removal of mch receptors from the plasma membrane was largely responsible for this process . surprisingly we found that mch receptors internalize very poorly in bhk-570 cells unless -arrestin 1 or -arrestin 2 is overexpressed . similarly , grk2 phosphorylation of mchr1 is thought to be important because a dominant - negative grk2 was able to eliminate even the small amount of receptor internalization induced by mch . we conclude that mch receptor signaling and desensitization are particularly sensitive to cellular levels of -arrestins and grk2 , which should be strongly taken under consideration when interpreting mch signaling studies across different cell types . perturbation of the cellular levels of these accessory proteins could greatly influence the activity of this appetite - stimulating pathway .
the regulation of appetite is complex , though our understanding of the process is improving . 
 the potential role for the melanin - concentrating hormone ( mch ) signaling pathway in the treatment of obesity is being explored by many . 
 it was hypothesized that internalization of mch receptors would act to potently desensitize cells to mch . despite potent desensitization of erk signaling by mch in bhk-570 cells 
 , we were unable to observe mch - mediated internalization of mch receptor 1 ( mchr1 ) by fluorescence microscopy . 
 a more quantitative approach using a cell - based elisa indicated only 15% of receptors internalized , which is much lower than that reported in the literature . 
 when -arrestins were overexpressed in our system , removal of receptors from the cell surface was facilitated and signaling to a leptin promoter was diminished , suggesting that internalization of mchr1 is sensitive to cellular -arrestin levels . 
 a dominant - negative grk construct completely inhibited loss of receptors from the cell surface in response to mch , suggesting that the internalization observed is phosphorylation - dependent . since desensitization of mch - mediated erk 
 signaling did not correlate with significant loss of mchr1 from the cell surface , we hypothesize that in this model system regulation of mch signaling may be the result of segregation of receptors from signaling components at the plasma membrane .