briefly , gst - tagged rat -arrestin1 in the pgex4 t vector was transformed into bl21(de3 ) cells , large scale expression cultures were grown in terrific broth , and induced with 1 mm iptg for 16 hr at 16 c .
cell pellets were lysed in 20 mm hepes ph 7.4 , 150 mm nacl , 1 mm pmsf and 2 mm dtt using a micro fluidizer , and the lysate was bound to glutathione sepharose at 4 c for 2 hr . beads were washed in lysis buffer and -arrestin1 was eluted by overnight incubation with thrombin at 4 c .
-arrestin1 was then purified with a hitrap q column and eluted by a linear gradient of nacl .
peak fractions were pooled and purified protein was dialyzed in 20 mm hepes ph 7.4 and 150 mm nacl .
the phage library was panned against biotinylated -arrestin1 bound to v2rpp and immobilized on streptavidin beads .
beads were washed three times and bound phages were amplified by infecting e. coli xl-1 blue cells .
amplified phage were precipitated and used for a second and third round of panning . to select against fabs that bind to the inactive conformation of -arrestin1
, beads coated with the -arrestin1:v2rpp complex were first incubated with phage and then with 1 m non - biotinylated -arrestin1 .
subsequently , phage were eluted with dithiothreitol ( dtt ) and resulting clones were used for single point elisa to test their selectivity towards -arrestin1 bound to v2rpp .
-arrestin1 was incubated with either non - phosphorylated v2 vasopressin peptide ( v2rnp ) or v2rpp in a 1:3 molar ratio for 30 min at 25 c . subsequently
, purified fabs were added at a 1:2 molar ratio with -arrestin1 and incubated for additional 30 min at 25 c .
then , pre - washed protein a beads ( pierce ) were added to the reactions and incubated for 30 min at 25 c .
beads were washed 4 times with 1 ml buffer ( 20 mm hepes ph 7.4 , 150 mm nacl ) and proteins were eluted with sds - page gel loading buffer .
fab30 displayed the greatest difference in its ability to co - immunoprecipitate -arrestin1 between v2rnp and v2rpp and was therefore chosen for further characterization ( reis & lefkowitz , manuscript in preparation ) .
sf9 insect cells were co - infected with baculovirus encoding an n - terminal flag tagged 2-v2r ( a chimeric receptor with 2ar residues 1 to 341 and v2 vasopressin receptor residues 328 through 372 ) and grk2-caax ( grk2 with a membrane tethering prenylation signal ) . following viral infection for 72 hours at 27 c ,
cells were incubated with 10 m isoproterenol at 37 c for 15 min to induce receptor phosphorylation .
membranes were extensively washed in order to remove isoproterenol used for receptor phosphorylation . for radioligand binding , membranes were incubated with 60 pm [ i ] cyanopindolol ( ge healthcare lifescience ) in radioligand binding buffer ( 50 mm tris , ph 7.4 , 50 mm potassium acetate , 0.5 mm magnesium chloride , 1 mm ascorbic acid ) with varying concentrations of freshly prepared isoproterenol .
binding reactions were performed in parallel , with 1 m -arrestin1 ( residues 1 - 393 ) incubated either in the presence or absence of 10 m fab30 .
binding reactions were incubated for 90 min at 27 c , followed by rapid harvesting on a gf - b filter and scintillation counting in a packard gamma counter .
competition binding data were analyzed by a non - linear curve - fitting procedure where low and high affinity values were computed globally using a two - site binding model ( graphpad prism ) .
the f - test was used to test whether fab30 significantly altered the amount of 2-v2r coupled to -arrestin .
purified , phosphorylated 2-v2r was prepared bound to the potent agonist 2ar agonist bi-167107 and incubated at a concentration of 1 m with 3 m -arrestin1 with and without fab30 at 25 c for 2 hours in a buffer comprised of 20 mm hepes , ph 7.4 , 150 mm nacl , 0.01% mng ( lauryl maltose neopentyl glycol ) .
beads were washed and protein was eluted with 5 mm edta and 0.25 mg / ml flag peptide and elution fractions were analyzed on a 4 - 20% sds - page gel and stained with coomassie .
-arrestin1 ( 20 m ) was incubated with v2rpp ( 27 m ) for 30 min at 25 c .
an excess of fab30 was added and the complex was incubated for 1 hour at 25 c .
the -arrestin1:v2rpp : fab30 complex was purified from excess fab30 and v2rpp by size exclusion chromatography in 20 mm hepes ph 7.5 , 150 mm nacl , and 1 mm tcep . the purified complex was concentrated to 8 mg / ml using a centrifugal concentrator ( vivaspin , ge healthcare ) .
crystals were grown in hanging drops containing 1 l of complex solution and 0.5 l of a well solution composed of 17% peg 3350 , 0.1 m hepes ph 7.5 , and 0.2 m l - proline .
drops were stored at 20 c and crystals appeared within 24 hours and grew to full size within 3 days ( fig .
crystals were flash frozen in liquid nitrogen after a 30 second soak in 19% peg 3350 , 0.1 m hepes ph 7.5 , 0.2 m l - proline , and 20% ethylene glycol .
diffraction data were collected at the advanced photon source gm / ca - cat beamline 23id - d . although typical crystals grew to over 300 m in two dimensions , and over 100 m in the third dimension , we utilized a 10 m - sized beam to collect multiple full datasets from the highest quality regions of the crystal .
a full dataset from the single best region of the crystal was indexed , integrated , and scaled with hkl-2000 .
the structure of the complex was solved by molecular replacement using phaser . due to the conformational changes
observed for -arrestin1 , it proved necessary to first search for fab30 ( pdb i d : 3eff ; fab2 , with the complementary determining regions omitted , was used as a search model for fab30 ) , followed by only the c - domain of -arrestin1 ( pdb i d : 1jsy ) . a subsequent search for the n - domain of -arrestin1 failed in multiple attempts ; the n - domain was then manually placed to fit the electron density .
a significant decrease in rfree upon rigid body refinement of the n - domain provided confidence in the final molecular replacement solution .
the resulting model was then iteratively refined by building regions of -arrestin1 , v2rpp , and fab30 in coot and refining in phenix .
we utilized translation libration screw - motion ( tls ) refinement with groups defined within phenix .
for the v2rpp , the amino - acid register was determined by the strong electron density resulting from electron - rich phosphates on phosphoserine and phosphothreonine residues . as shown in fig .
s7 , the electron density for the v2rpp was clear , permitting confident placement of most side chains .
we used molprobity to assess statistics for the final model of the -arrestin1:v2rpp : fab30 complex . supplementary table 1 outlines statistics for data collection and refinement .
briefly , gst - tagged rat -arrestin1 in the pgex4 t vector was transformed into bl21(de3 ) cells , large scale expression cultures were grown in terrific broth , and induced with 1 mm iptg for 16 hr at 16 c .
cell pellets were lysed in 20 mm hepes ph 7.4 , 150 mm nacl , 1 mm pmsf and 2 mm dtt using a micro fluidizer , and the lysate was bound to glutathione sepharose at 4 c for 2 hr . beads were washed in lysis buffer and -arrestin1 was eluted by overnight incubation with thrombin at 4 c .
-arrestin1 was then purified with a hitrap q column and eluted by a linear gradient of nacl .
peak fractions were pooled and purified protein was dialyzed in 20 mm hepes ph 7.4 and 150 mm nacl .
the phage library was panned against biotinylated -arrestin1 bound to v2rpp and immobilized on streptavidin beads .
beads were washed three times and bound phages were amplified by infecting e. coli xl-1 blue cells .
amplified phage were precipitated and used for a second and third round of panning . to select against fabs that bind to the inactive conformation of -arrestin1
, beads coated with the -arrestin1:v2rpp complex were first incubated with phage and then with 1 m non - biotinylated -arrestin1 .
subsequently , phage were eluted with dithiothreitol ( dtt ) and resulting clones were used for single point elisa to test their selectivity towards -arrestin1 bound to v2rpp .
-arrestin1 was incubated with either non - phosphorylated v2 vasopressin peptide ( v2rnp ) or v2rpp in a 1:3 molar ratio for 30 min at 25 c . subsequently
, purified fabs were added at a 1:2 molar ratio with -arrestin1 and incubated for additional 30 min at 25 c .
then , pre - washed protein a beads ( pierce ) were added to the reactions and incubated for 30 min at 25 c .
beads were washed 4 times with 1 ml buffer ( 20 mm hepes ph 7.4 , 150 mm nacl ) and proteins were eluted with sds - page gel loading buffer .
fab30 displayed the greatest difference in its ability to co - immunoprecipitate -arrestin1 between v2rnp and v2rpp and was therefore chosen for further characterization ( reis & lefkowitz , manuscript in preparation ) .
sf9 insect cells were co - infected with baculovirus encoding an n - terminal flag tagged 2-v2r ( a chimeric receptor with 2ar residues 1 to 341 and v2 vasopressin receptor residues 328 through 372 ) and grk2-caax ( grk2 with a membrane tethering prenylation signal ) . following viral infection for 72 hours at 27 c
, cells were incubated with 10 m isoproterenol at 37 c for 15 min to induce receptor phosphorylation .
membranes were extensively washed in order to remove isoproterenol used for receptor phosphorylation . for radioligand binding , membranes were incubated with 60 pm [ i ] cyanopindolol ( ge healthcare lifescience ) in radioligand binding buffer ( 50 mm tris , ph 7.4 , 50 mm potassium acetate , 0.5 mm magnesium chloride , 1 mm ascorbic acid ) with varying concentrations of freshly prepared isoproterenol .
binding reactions were performed in parallel , with 1 m -arrestin1 ( residues 1 - 393 ) incubated either in the presence or absence of 10 m fab30 .
binding reactions were incubated for 90 min at 27 c , followed by rapid harvesting on a gf - b filter and scintillation counting in a packard gamma counter .
competition binding data were analyzed by a non - linear curve - fitting procedure where low and high affinity values were computed globally using a two - site binding model ( graphpad prism ) .
the f - test was used to test whether fab30 significantly altered the amount of 2-v2r coupled to -arrestin .
purified , phosphorylated 2-v2r was prepared bound to the potent agonist 2ar agonist bi-167107 and incubated at a concentration of 1 m with 3 m -arrestin1 with and without fab30 at 25 c for 2 hours in a buffer comprised of 20 mm hepes , ph 7.4 , 150 mm nacl , 0.01% mng ( lauryl maltose neopentyl glycol ) .
beads were washed and protein was eluted with 5 mm edta and 0.25 mg / ml flag peptide and elution fractions were analyzed on a 4 - 20% sds - page gel and stained with coomassie .
-arrestin1 ( 20 m ) was incubated with v2rpp ( 27 m ) for 30 min at 25 c .
an excess of fab30 was added and the complex was incubated for 1 hour at 25 c .
the -arrestin1:v2rpp : fab30 complex was purified from excess fab30 and v2rpp by size exclusion chromatography in 20 mm hepes ph 7.5 , 150 mm nacl , and 1 mm tcep . the purified complex was concentrated to 8 mg / ml using a centrifugal concentrator ( vivaspin , ge healthcare ) .
crystals were grown in hanging drops containing 1 l of complex solution and 0.5 l of a well solution composed of 17% peg 3350 , 0.1 m hepes ph 7.5 , and 0.2 m l - proline .
drops were stored at 20 c and crystals appeared within 24 hours and grew to full size within 3 days ( fig .
crystals were flash frozen in liquid nitrogen after a 30 second soak in 19% peg 3350 , 0.1 m hepes ph 7.5 , 0.2 m l - proline , and 20% ethylene glycol .
diffraction data were collected at the advanced photon source gm / ca - cat beamline 23id - d .
although typical crystals grew to over 300 m in two dimensions , and over 100 m in the third dimension , we utilized a 10 m - sized beam to collect multiple full datasets from the highest quality regions of the crystal .
a full dataset from the single best region of the crystal was indexed , integrated , and scaled with hkl-2000 .
the structure of the complex was solved by molecular replacement using phaser . due to the conformational changes
observed for -arrestin1 , it proved necessary to first search for fab30 ( pdb i d : 3eff ; fab2 , with the complementary determining regions omitted , was used as a search model for fab30 ) , followed by only the c - domain of -arrestin1 ( pdb i d : 1jsy ) . a subsequent search for
the n - domain of -arrestin1 failed in multiple attempts ; the n - domain was then manually placed to fit the electron density . a significant decrease in rfree upon rigid body refinement of the n - domain provided confidence in the final molecular replacement solution .
the resulting model was then iteratively refined by building regions of -arrestin1 , v2rpp , and fab30 in coot and refining in phenix .
we utilized translation libration screw - motion ( tls ) refinement with groups defined within phenix .
for the v2rpp , the amino - acid register was determined by the strong electron density resulting from electron - rich phosphates on phosphoserine and phosphothreonine residues . as shown in fig .
s7 , the electron density for the v2rpp was clear , permitting confident placement of most side chains .
we used molprobity to assess statistics for the final model of the -arrestin1:v2rpp : fab30 complex .
 the functions of g - protein coupled receptors ( gpcrs ) are primarily mediated and modulated by three families of proteins : the heterotrimeric g proteins , the g - protein coupled receptor kinases ( grks ) , and the arrestins1 . 
 g proteins mediate activation of second messenger - generating enzymes and other effectors , grks phosphorylate activated receptors2 , and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization3 . 
 arrestins activated by interaction with phosphorylated receptors can also mediate g protein - independent signaling by serving as adaptors to link receptors to numerous signaling pathways4 . despite their central role in regulation and signaling of gpcrs , a structural understanding of -arrestin activation and interaction with 
 gpcrs is still lacking . here , we report the crystal structure of -arrestin1 in complex with a fully phosphorylated 29 amino acid carboxy - terminal peptide derived from the v2 vasopressin receptor ( v2rpp ) . 
 this peptide has previously been shown to functionally and conformationally activate -arrestin15 . to capture this active conformation 
 , we utilized a conformationally - selective synthetic antibody fragment ( fab30 ) that recognizes the phosphopeptide - activated state of -arrestin1 . 
 the structure of the -arrestin1:v2rpp : fab30 complex shows striking conformational differences in -arrestin1 compared to its inactive conformation . 
 these include rotation of the amino and carboxy - terminal domains relative to each other , and a major reorientation of the  lariat loop  implicated in maintaining the inactive state of -arrestin1 . 
 these results reveal , for the first time at high resolution , a receptor - interacting interface on -arrestin , and they suggest a potentially general molecular mechanism for activation of these multifunctional signaling and regulatory proteins . 
ONLINE METHODS
Purification of -arrestin1
Selection and characterization of Fab
Selectivity of Fabs towards V
Radioligand binding
Effect of Fab30 on 
Preparation of -arrestin1:V
Supplementary Material