Báo cáo khoa học: The role of helix 8 and of the cytosolic C-termini in the internalization and signal transduction of B1 and B2 bradykinin receptors potx

The role of helix and of the cytosolic C-termini in the internalization and signal transduction of B1 and B2 bradykinin receptors Alexander Faussner1, Alexandra Bauer1, Irina Kalatskaya1, Steffen Schussler1, Cornelia Seidl1, ă David Proud2 and Marianne Jochum1 Ludwig-Maximilians-Universitat, Abteilung fur Klinische Chemie und Klinische Biochemie, Munchen, Germany ă ¨ ¨ Department of Physiology & Biophysics, University of Calgary, Alberta, Canada Keywords G protein-coupled receptor, helix modeling, internalization, receptor chimera Correspondence A Faussner, Ludwig-MaximiliansUniversitaet Muenchen, Abt Klinische Chemie und Klinische Biochemie, Nussbaumstr 20, D-80336 Muenchen, Germany Tel: +49 89 51602602 Fax: +49 89 51604740 E-mail: alexander.faussner@ med.uni-muenchen.de (Received 21 July 2004, revised 14 September 2004, accepted 15 September 2004) doi:10.1111/j.1432-1033.2004.04390.x Determinants for desensitization and sequestration of G protein-coupled receptors often contain serine or threonine residues located in their C-termini The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown Mutagenesis studies with the B2 bradykinin receptor (B2wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including I347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist-inducible internalization and the prevention of receptor hypersensitivity Chimeras of the noninternalizing B1 bradykinin receptor (B1wt) containing these B2wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required In fact, further substitution of the B1wt C-terminus with corresponding B2wt regions either at C330(7.71) following putative helix (B1CB2) or at the preceding Y312(7.53) in the NPXXY sequence (B1YB2) resulted in chimeras displaying rapid internalization Intriguingly, however, exchange performed at K322(7.63) within putative helix generated a slowly internalizing chimera (B1KB2) Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B1wt to serine (as in B2wt) as being responsible for this effect The slowly internalizing chimera as well as a B1wt point-mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B1wt or the other chimeras The slow internalization of B1KB2 was also accompanied by a lack of agonistinduced phosphorylation, that in contrast was observed for B1YB2 and B1CB2, suggesting that putative helix is either directly or indirectly (e.g via G protein activation) involved in the interaction between the receptor and receptor kinases G protein-coupled receptors (GPCRs) form a vast and diverse superfamily of proteins with seven transmembrane-spanning domains They transduce specific external stimuli to intracellular second messenger-dependent effector cascades via recruitment and activation of heterotrimeric G proteins [1] To protect cells from chronic overstimulation, desensitization processes such as the rapid attenuation of receptor responsiveness and Abbreviations BK, bradykinin; Bxwt, wild-type Bx bradykinin receptor; DAK, desArg10kallidin; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HEK, human embryonic kidney; IP, inositol phosphate FEBS Journal 272 (2005) 129–140 ª 2004 FEBS 129 Role of helix and C-termini in bradykinin receptors G protein uncoupling are essential Some of these desensitization mechanisms involve the translocation of the stimulated receptor to distinct compartments and endocytosis after phosphorylation of serine ⁄ threonine residues mostly located in the receptor C-termini (reviewed in [2]) Little is known so far about the sequence context in which these residues have to appear to become phosphorylated by kinases and to be recognized by the internalization machinery In particular, the receptor specificity of these motifs is not completely understood The B1 bradykinin receptor (B1wt) is one of the few receptors belonging to the class A family of rhodopsin-like ⁄ b2-adrenergic-like GPCRs that does not get internalized, i.e sequestered to intracellular compartments upon agonist stimulation [3] It does, however, respond with translocation to caveolae but these remain essentially on the cell surface [4,5] No phosphorylation of B1wt either under basal conditions or after stimulation has been detected [6] The B2 bradykinin receptor (B2wt), by contrast, is a more typical GPCR that gets internalized rapidly following activation Phosphorylation of several serine ⁄ threonine residues in the C-terminus of this receptor, and the importance of these events for receptor sequestration, have been described in detail [7] Whether coupling of b-arrestin(s) then follows this, and whether internalization occurs via clathrin-coated pits, caveolae or other less well-defined mechanisms is still a topic of debate [5,7,8] The two bradykinin (BK) receptor subtypes exhibit a relatively low overall amino acid identity of about 36% [9,10], most of it located in the transmembrane regions Both receptors stimulate phospholipase Cb-mediated inositol phosphate (IP) release leading to an elevation of intracellular [Ca2+] levels, primarily via coupling to G protein Gq ⁄ 11 [3,10,11] They become activated by the kinins, small proinflammatory peptides with great vasoactive potential implicated as mediators of inflammation, pain and hyperalgesia [12,13] The nonapeptide BK and Lys-BK (kallidin) bind with high affinity to B2wt but not B1wt Removal of the C-terminal arginine through carboxypeptidases generates desArg9-bradykinin and desArg10kallidin (DAK), two peptides that now bind exclusively to the B1wt [14] In this study we wanted to exploit the fact that the B1wt does not internalize as part of a gain-of-function approach to provide insight into the receptor specificity of the B2wt internalization motif The resulting data also hint at a receptor specific role of the putative helix in G protein activation and interaction with receptor kinases 130 A Faussner et al Results Construction of truncated and point mutated B2wts and B1 ⁄ B2 receptor chimeras Several studies with truncations of, and deletions in, the C-terminal part of B2wt have demonstrated that this part plays a central role in the internalization of this receptor [7,15,16] A similar function of the C-terminus was also observed in other GPCRs with short third intracellular loops [2] In particular, several serine or threonine residues that become phosphorylated by protein kinase C and ⁄ or by GPCR kinases (GRKs) following receptor activation are absolutely required for rapid B2wt sequestration [17] To determine the C-terminal sequence(s) of the B2wt minimally required for internalization we created two new B2wt truncations, I347* and N338* (Fig 1) The former removed the C-terminus including residue S348, which has been shown to be responsible for the basal phosphorylation of the B2wt, while the latter truncation deleted all serine and threonine residues (S339, T342, T345, S346) shown to be phosphorylated following stimulation of the receptor [17] In addition, a triple alanine replacement of T345-S346-I347(S348) (mutated residues are underlined) was made, as this sequence strongly resembles the C-terminal STLSmotif in the AT1A angiotensin II receptor, where a triple alanine substitution of STL almost completely abolished receptor sequestration [18] All of these B2 receptor constructs were highly expressed (Table 1) We took care therefore to use [3H]BK concentrations below 1.5 nm, as we have shown that receptor internalization rates are independent of agonist concentration in this range [19] The truncation I347* internalized as rapidly as B2wt (Fig 2A) demonstrating that the distal C-terminus, and in particular S348 and its basal phosphorylation, not play a decisive role in the sequestration process This notion was further supported by results obtained with a point mutation of S348 to alanine that exhibited an almost identical internalization rate as the B2wt ([7] and data not shown) In contrast, deletion of all phosphorylation sites in N338* led to an extremely diminished [3H]BK internalization (Fig 2A) Indeed, even the internalization calculated for each time point is an overestimate because a shift to lower affinity at 37 °C by the receptors remaining on the cell surface can be assumed, as there was a clear drop in surface binding that could not be accounted for by the amount of internalized agonist [20] As the internalization is expressed in percentage of total binding, decreasing the binding affinity of the FEBS Journal 272 (2005) 129–140 ª 2004 FEBS A Faussner et al Role of helix and C-termini in bradykinin receptors Fig Schematic representation of the C-terminal B1wt and B2wt sequences and chimera thereof The C-terminal sequences beginning at transmembrane domain are shown B1wt parts are indicated in filled circles, B2wt portions in unfilled ones The phosphorylation sites in B2wt are highlighted in light grey, and the position number is indicated The grey box outside the membrane indicates the region of the putative cytosolic helix as found in the crystal structure of bovine rhodopsin [24] The assumed palmitoylation of B1wt and B2wt is indicated surface receptors simulates an apparent increase in internalization over time Although it internalized [3H]BK much slower than the B2wt, N338* nevertheless was able to induce an accumulation of total IPs identical to that observed for the B2wt (Table 1) This truncated receptor even became hypersensitive, as its EC50 for the IP response was 10-fold lower than that of B2wt (0.072 ± 0.038 nm vs 0.79 ± 0.34 nm; Table 1) Most interestingly, the effects of a truncation at N338 could also be achieved in part by the triple mutation TSIfiAAA as this construct displayed similar properties to truncation N338* It exhibited a markedly reduced capacity to internalize [3H]BK albeit not as diminished as truncation N338* and was at least as hypersensitive with an EC50 ¼ 0.058 ± 0.06 nm (Table 1) This sequence obviously contributes significantly to agonist internalization and signaling of B2wt FEBS Journal 272 (2005) 129–140 ª 2004 FEBS However, transfer of the B2wt C-terminus starting with this sequence, to the C-terminus of the intact noninternalizing B1wt (B1RB2; Fig 1), conferred very little capability to internalize its agonist to the B1wt (Fig 2B) The chimera B1NB2 containing all serine and threonine residues critical for B2wt sequestration, in contrast, was able to internalize [3H]DAK at a rate approximately half of the maximal rate (40% after 10 min) seen for the B2wt with [3H]BK (Fig 2B) As it was obviously not sufficient to simply add the B2wt phosphorylation sites to the B1wt to gain full receptor sequestration as observed in the B2wt, we further substituted the C-termini of the B2wt into the B1wt at two residues conserved in both receptor subtypes (Fig 1); specifically at the conserved cysteine [Cys330(7.71) in B1wt, Cys324(7.72) in B2wt] that in the B2wt is palmitoylated (chimera B1CB2) and at Y7.53 within the NPXXY sequence (chimera B1YB2) 131 Role of helix and C-termini in bradykinin receptors A Faussner et al Table Receptor density (Bmax), receptor affinity (Kd), basal and stimulated total IP accumulation, and EC50 of B2wt, B1wt and B1 ⁄ B2 receptor chimera ND, not determined IP accumulation Receptor construct Bmaxa (fmolỈmg protein)1) Kd (nM) Unstimulated (30 minỈbasal)1) EC50 ± SEM (nM) B2wt I347* TSIfiAAA N338* B1wt B1RB2 B1NB2 B1CB2 B1KB2 B1KB2 ⁄ SfiV B1KB2 ⁄ QGVfiKQ B1KB2 ⁄ VCfiCV B1YB2 B1V323S 10400 5020 5298 3832 625 127 511 1701 1823 1758 2142 1786 2957 846 3.91 ± 1.06 3.76 ± 1.61 2.82 ± 0.92 4.03 ± 0.80 1.11 ± 0.12 1.09 ± 0.11 ND 1.48 ± 0.17 ND 1.59 ± 0.44 ND ND 1.85 ± 1.4 ND 1.93 ± 0.17 ND 2.29 ± 0.77 2.54 ± 0.38 1.6 ± 0.2 ND ND 1.53 ± 0.14 1.84 ± 0.25 1.31 ± 0.12 1.33 ± 0.12 1.42 ± 0.06 1.50 ± 0.15 1.44 ± 0.08 12.86 ± 1.37 (n ¼ 7) ND 10.68 ± 2.25 (n ¼ 3) 13.57 ± 1.76 (n ¼ 3) 8.41 ± 0.52 (n ¼ 7) ND ND 7.5 ± 0.6 (n ¼ 7) 4.1 ± 0.2b (n ¼ 5) 7.2 ± 0.8 (n ¼ 3) 4.6 ± 0.9b (n ¼ 3) 4.3 ± 0.1b (n ¼ 3) 8.7 ± 0.8 (n ¼ 6) 4.59 ± 0.84b (n ¼ 3) 0.79 ± 0.34 (n ¼ 4) 1.13 ± 0.47 (n ¼ 4) 0.058 ± 0.006 (n ¼ 3) 0.072 ± 0.038 (n ¼ 3) 0.37 ± 0.06 (n ¼ 7) ND 0.28 ± 0.1 (n ¼ 3) 1.0 ± 0.08 (n ¼ 3) 0.7 ± 0.3 (n ¼ 3) 1.7 ± 0.2 (n ¼ 3) 2.0 ± 0.2 (n ¼ 3) 0.8 ± 0.1 (n ¼ 3) 2.2 ± 0.2 (n ¼ 3) 0.35 ⁄ 0.28 a ± ± ± ± ± ± ± ± ± ± ± ± ± ± Estimated with 10 nM [3H]DAK b 600 900 1080 290 24 24 160 503 664 150 623 320 1041 128 P < 0.001 vs B1wt at the end of the seventh transmembrane domain We have shown previously that a B1CB2 chimera stably expressed in Chinese hamster ovary cells was sequestered rapidly upon activation [16] This was confirmed in human embryonic kidney (HEK) 293 cells (Fig 2B) As the chimera B1YB2 exhibited a slightly attenuated internalization compared to B1CB2 (Fig 2B), and the latter apparently did not gain the full internalization capability of the B2wt, we next tested the possibility that there is an optimum site for creating rapidly internalizing chimeras at K7.63 between these two residues and generated the chimera B1KB2 (Fig 1) Surprisingly, B1KB2 showed poor ability to internalize [3H]DAK (30% after 10 min), with an internalization far below those seen for B1CB2 and B1YB2 (Fig 2B) test these possibilities: (a) B1KB2 ⁄ QGVfiKQ; (b) B1KB2 ⁄ VCfiCV; and (c) B1KB2 ⁄ SfiV (Fig 3A) Substituting KQ for QGV in B1KB2 led to distinctly increased agonist internalization as compared with B1KB2 This increase was not due to a corrected position of the cysteine, as it was not observed with B1KB2 ⁄ VCfiCV (Fig 3B) A major effect, however, was seen with the change of the polar serine (back) to the nonpolar valine (B1KB2 ⁄ SfiV), the amino acid that is normally found in this position in the B1wt This replacement led to a chimera exhibiting rapid internalization (60% after 10 min) that was comparable to that of B1CB2 and B1YB2 (Fig 2B) Agonist-induced internalization of modified B1KB2 constructs Phosphorylation patterns of B2wt and of B1 ⁄ B2 chimeras reflect their agonist-inducible internalization The segment between the NPXXY motif and the conserved cysteine represents one of the regions with the highest sequence identity between B1wt and B2wt The different internalization of B1KB2 and B1CB2 was therefore even more surprising given that these two chimeras have only minor sequence differences (Fig 3A) Therefore we considered three possibilities to explain the cause of this drop in the internalization of B1KB2 as compared to B1CB2 First, that the two residues (KQ) preceding the cysteine were pivotal; second, that the cysteine itself needs to be at a specific position in the C-terminus; or third, that the B1 residue V323 instead of the serine is essential in this position Thus, we created three additional chimeras to Agonist-induced phosphorylation of serine and threonine residues in the C-terminus has been shown to be a prerequisite for internalization of B2wt and other receptors [17,21] B2wt in HEK 293 cells displayed a distinct phosphorylation even in the absence of an agonist (Fig 4), as reported recently [22] When stimulated for with a saturating concentration of lm BK at 37 °C, however, B2wt responded with a marked increase (2.50 ± 0.15-fold over basal) in phosphorylation The chimera on the other hand displayed little basal phosphorylation in the absence of their agonist DAK, although this may, in part, be a sensitivity problem due to their lower expression levels Nevertheless, the rapidly internalizing chimeras B1YB2 and B1CB2 132 FEBS Journal 272 (2005) 129–140 ª 2004 FEBS A Faussner et al Role of helix and C-termini in bradykinin receptors 100 Internalization [% of total] A I347* 80 B2 wt 60 TSI->AAA 40 20 N338* 0 10 15 20 25 30 Time [min] 100 Internalization [% of total] B 80 B1 CB2 B1 YB2 60 B1 NB2 40 B1 KB2 20 B1 RB2 B1 wt 0 10 15 Time [min] 20 25 30 Fig Internalization of [3H]agonist by wild-type bradykinin receptors, truncations and chimera HEK 293 cells expressing the wildtype receptors B1wt or B2wt, chimera thereof, or B2wt truncations or mutations were preincubated with the appropriate[3H] agonist: (A) < 1.5 nM [3H]BK; (B) nM[3H]DAK) for 90 on ice Internalization was started by placing the cells in a 37 °C water bath and stopped at the indicated times Surface-bound and internalized agonist were determined as described in Material and methods Agonist internalization was expressed as percentage of total bound agonist Results are given as mean ± SEM of at least three independent experiments performed in triplicate responded to stimulation with lm DAK with a distinct increase in phosphorylation The slowly internalizing B1KB2, in contrast, exhibited no significant phosphorylation even when challenged with DAK Total IP accumulation of B1wt and B1 ⁄ B2 chimeras parallels their agonist-inducible internalization The IP release was expressed as unstimulated or DAKstimulated accumulation of total IPs for 30 at 37 °C compared to the IP content of control cells that had remained at °C There was a clear correlation between the agonist-inducible internalization and the IP accumulation it could induce when stimulated (Fig 5) All chimeric constructs displaying rapid agonist-inducible internalization (B1CB2, B1YB2, B1KB2 ⁄ FEBS Journal 272 (2005) 129–140 ª 2004 FEBS Fig [3H]DAK internalization of B1KB2 derived constructs (A) Alignment of the relevant sequences of the B1CB2 and B1KB2-derived chimera compared to wild-type bradykinin receptor subtypes Residues found in B1wt are in capital letters; those found in B2wt are in lowercase Amino acids identical to the B1wt sequence are indicated by dashes The residues mutated in B1KB2 are in bold To allow comparison the sequence of rhodopsin is also shown (B) Internalization of [3H]DAK was performed as described in the legend to Fig Each time point represents the mean ± SEM of at least three different experiments done in triplicate SfiV) showed an IP response similar to that seen for B1wt (8.41 ± 0.52 fold for B1wt and 7.2–8.7-fold for the chimera) In contrast, the chimera that internalized poorly (B1KB2, B1KB2 ⁄ QGVfiKQ, B1KB2 ⁄ VCfiCV) showed a significantly reduced IP signal (4.1–4.6-fold) despite the fact that they were expressed at similar levels to the chimeras that became rapidly internalized (Table 1) These results suggested that V323 might play a role in the activation of phospholipase C through B1wt Indeed, exchange of V323 for a serine in B1wt (construct B1 V323S) resulted in a clearly reduced IP response (5.28 ± 0.91 vs 8.41 ± 0.52 for B1wt; Table and Fig 5) Discussion Phosphorylation of serine or threonine residues in the C-terminus of GPCRs by second messenger kinases or specific GRKs is a requirement for receptor sequestration [23] However, the context in which these residues have to appear, or the receptor specificity of their function is not very well understood 133 Mr 75 12 Total inositol phosphate release/control B1CB2 A Faussner et al B1KB2 B1YB2 B2wt control Role of helix and C-termini in bradykinin receptors unstimulated stimulated 10 *** 150 100 50 + - + - + - + - + Fig Agonist-induced phosphorylation of B2wt and B1 ⁄ B2-chimera Upper panel: HEK293 cells expressing B2wt, B1YB2, B1KB2, or B1CB2 were labeled for 10 h with [32P]orthophosphate before stimulation with lM BK and lM DAK, respectively, for Cells were lysed and proteins were solubilized, immunoprecipitated and visualized by autoradiography Molecular size markers are indicated to the left Lower panel: protein phosphorylation, given as optical densities of the bands in the area between 50 and 85 kDa, is presented as mean ± SD from three independent experiments; unstimulated B2wt was set as 100% The bradykinin receptor subtypes are an excellent tool to address this issue using both loss- and gain-offunction approaches, as B2wt gets internalized rapidly following stimulation whereas B1wt does not become sequestered [3] As both receptors couple preferentially to the same Ga subunit (Gq ⁄ 11) differential signaling is less likely to explain differences in internalization than in two receptors signaling through different G proteins Internalization patterns of truncations I347* and N338*, and the triple point mutant TSIfiAAA more closely defined the sequence necessary for the internalization of B2wt Because I347* was internalized as rapidly as B2wt, while the TSIfiAAA mutant showed reduced internalization and N338* almost none, the nine residues from S339 to I347 (SMGTLRTSI) must play a key role in B2wt sequestration The following results from our gain-of-function approach, however, led us to conclude that additional motifs in the more B V V C KB B /V KB B 2 /S C KQ KB B 200 KB /Q G V B YB B B C B 250 BK (DAK) - t w B % of Basal Phosphorylation 300 V3 23 S 50 134 *** *** *** Fig Total IP accumulation of B1wt and chimera HEK293 cells expressing the indicated receptor constructs were preincubated with 50 mM LiCl, and then with (stimulated) or without (unstimulated) lM DAK for 90 on ice IP accumulation was started in a water bath at 37 °C and stopped after 30 as described in Materials and methods The basal IP accumulation level was determined on ice The results are expressed as fold total IP accumulation above basal and given as mean ± SEM of at least three different experiments performed in triplicate proximal portion of the C-terminus also play a role in receptor internalization First, transfer of the B2wt C-terminus starting with the nine residues containing all known B2wt phosphorylation sites did not permit maximal internalization of [3H]DAK, indicating that this nine residue sequence is either receptor specific or that other motifs must contribute to B2wt sequestration Second, faster internalization was obtained when more extended parts of the C-terminus beginning either at conserved C7.71(B1) ⁄ 7.72(B2) (B1CB2) or conserved Y7.53 (B1YB2) were transferred, indicating sequestration motifs in the region between the palmitoylated C324 and N338 Candidates would include G328-C329 and ⁄ or the negatively charged residues E332 and E337, as they are highly conserved in B2wt among species The chimera B1YB2 showed a slightly lower internalization compared to B1CB2 We therefore tested whether there was an optimum chimeric exchange point between these two mutation sites Intriguingly, exchange at a conserved lysine (K7.68) between these two sites resulted in a poorly internalizing chimera (B1KB2, Fig 2B) The crystal structure of inactive bovine rhodopsin [24] suggested an explanation for this result by revealing an additional helix close to the seventh transmembrane domain with a cytosolic localization parallel to the cell membrane Structure prediction programs [25] indicated that both B1wt and B2wt may also contain a helix Our results show that chiFEBS Journal 272 (2005) 129–140 ª 2004 FEBS A Faussner et al meric receptors with a helix derived either completely from B1wt (B1CB2) or B2wt (B1YB2) were internalized rapidly whereas a receptor with a chimeric helix (B1KB2) was internalized slowly As the latter displayed no agonist-induced phosphorylation – in contrast to chimera B1YB2 – this is probably caused by an impaired interaction with, or activation of, receptor kinases resulting in the observed slow internalization Further examination of helix revealed that S316 in the B2wt sequence of B1KB2 is responsible for the slow sequestration of this chimera (Fig 3B) Helices of the two receptor subtypes show different charge distributions despite their high sequence identity (Fig 6) The B2wt exhibits a highly charged N-terminal half (two arginines and three lysines) but due to S316 does not display a clear amphipathic structure The N-terminal half of B1wt, by contrast, is less positively charged (two arginines and one lysine) but B1wt has a strict amphipathic arrangement of the amino acid residues This arrangement is probably important in Role of helix and C-termini in bradykinin receptors receptor signaling because: (a) interruption of the amphipathic structure of B1wt helix in B1wt and B1KB2 through the presence of a serine in position of V323 leads to a strong attenuation of the IP signal; (b) substitution of this serine with valine in B1KB2 fully recovers the IP signal; and (c) sequence alignment of all known B1 bradykinin receptors shows that hydrophobicity in this position is absolutely conserved, while this is not the case for the residues further downstream It has been reported recently that truncation of the B1wt C-terminus at T327 resulted in an 85% reduction of IP generation, whereas further stepwise truncation up to R320 – thus including removal of V323 – did not lead to any further decrease [26] Thus, it appears that the presence of a hydrophobic residue at position 323 in B1wt is not necessary for Gq ⁄ 11 activation but rather that a polar serine there interferes with this process This group also described a strongly increased basal activity for a B2 receptor construct where several C-terminal serine and threonine residues Fig Structural comparison of helix in B1wt, B2wt and chimera Helix (N-terminus on the left hand side) from both bradykinin receptor subtypes was modeled along the structure of bovine rhodopsin by means of DEEPVIEW ⁄ Swiss-PdbViewer v3.7 [34] The dark green ribbon presentation belongs to B1wt, light green ribbon-parts to B2wt The residues different in B1wt and B2wt are indicated in larger bold labels Basic amino acid residues are in blue, acidic residues in red, polar residues are yellow, and unpolar residues are colored in grey The black lines in B1KB2 and B1KB2 ⁄ SfiV show the transition between B1wt and B2wt in the chimera FEBS Journal 272 (2005) 129–140 ª 2004 FEBS 135 Role of helix and C-termini in bradykinin receptors were substituted by alanines [27] We also observed a tendency to increased basal activity in B2 constructs that were lacking all or some of these residues, i.e TSIfiAAA and N338*, which was significant only for the latter (P < 0.006) when compared to B2wt (Table 1) Much more apparent, however, was that, in our hands, these two constructs were hypersensitive, displaying an EC50 value that was more than 10-fold lower than that observed for B2wt (Table 1) As their Kd values were not significantly different this indicates that apparently relatively few receptors have to be occupied to achieve half-maximal stimulation It is important, of course, to keep in mind that the Kd was determined at °C where coupling to G proteins does not play a role, whereas the EC50 was obtained by determining the IP accumulation after 30 at 37 °C Nevertheless, it is likely that this hypersensitivity is related to the fact that the mutated residues play an important role in the internalization (Fig 2A) and in the desensitization of B2wt [17] Much lower BK concentrations than with B2wt may therefore be sufficient to activate enough receptors for half-maximal IP accumulation In our experiments, we did not observe a strong constitutive B1wt signaling activity as compared to B2wt, nor any significant differences between the B1wt and B1 ⁄ B2 chimera in terms of basal activity (Table 1) as was reported recently [26] This discrepancy may be due to different cell culture conditions (e.g use of horse serum vs fetal bovine serum), or to their transient low expression vs our stably high expression and renders difficult the comparability of our data Several reports indicate that a fourth cytoplasmic loop, formed by membrane insertion of a conserved palmitoylated cysteine, and in particular the part comprising putative helix 8, may be involved in the interaction of GPCRs with cognate G proteins Synthetic peptides from the C-terminus of the Ga subunit Gt and of the Gc subunit of transducin interacted with rhodopsin and kept it in an activated state [28] This interaction, however, was abolished in mutants with replacements in helix 8, suggesting that G protein subunits interact directly or indirectly with helix In other experiments, peptides with the sequence of helix of rhodopsin inhibited activation of Gt by rhodopsin [29] In the angiotensin II receptor AT1A point mutations in the region of putative helix abolished release of inositol trisphosphates and the GTP-inducible shift in receptor affinity In addition, peptides based on its helix sequence stimulated binding of GTPcS to Gq ⁄ 11 [30] All of these data point to an involvement of putative helix in the interaction with cognate G proteins As both bradykinin receptors coupled to the same Ga subunit Gq ⁄ 11 the different IP 136 A Faussner et al responses obtained with the wild-type receptors and the chimera let us speculate that each wild-type helix may be specific either for selected bc subunits or for either Gq or G11 Additional experimental work will be necessary to test this hypothesis, particularly as the two receptors, while both coupling to Gq ⁄ 11 (and Gi) may very well differ in their capability to activate other additional signaling pathways These potential differences in, for example, the transactivation of growth hormone receptors and in the activation of MAPK cascades, as well as different localizations of the receptor constructs before and after activation may also contribute to the observed results Although helix initially was found in the crystal structure of inactive bovine rhodopsin [24], prior studies using NMR and circular dichroism of peptides taken from the fourth cytoplasmic loop of the angiotensin II AT1A receptor also indicated that, under certain experimental conditions, an amphipathic a-helix was formed in this region [31] By contrast, NMR studies of peptides representing the same region of rhodopsin in membrane and detergent-free solutions displayed a different structure, with transmembrane domain being extended and the C-terminus up to the cysteine existing as a loop [32] Krishna et al [33] demonstrated that the environment in which the peptide exists determines its structure, and suggested that this region serves as a membrane recognition site because the presence of detergent or membrane lipids influences the formation of a helical structure These authors proposed that activation of the receptor, and subsequently of the G protein, leads to a change in the environment of helix resulting in the loss of the helical structure Mutation of specific residues in their model led to a strongly reduced propensity for helical formation with the N-terminus of helix being more influential than the C-terminal portion Based on this model, we could speculate that the two bradykinin receptor subtypes, and those chimeras with an intact ⁄ homogenous helix 8, are able to appropriately switch conformation, whereas the receptors with a chimeric helix have lost this capacity Taken together, our results demonstrate that almost full capability for receptor internalization can be conferred to the normally noninternalizing B1wt, via transfer of the C-terminus of B2wt, provided that the new chimeric receptors have an intact ⁄ homogeneous helix either from B2wt or B1wt or a chimeric B1 ⁄ B2 helix with a conserved V323 Chimeric receptors with a heterogeneous helix exhibited an identical effect on signaling as well as on internalization, i.e poor signaling was accompanied by reduced internalization We suggest therefore that helix is directly or indirectly FEBS Journal 272 (2005) 129–140 ª 2004 FEBS A Faussner et al involved in the interaction with receptor kinases and in receptor specific G protein activation Materials and methods Materials Flp-In T-REx (HEK 293) cells were purchased from Invitrogen (Groningen, the Netherlands) and [2,3-prolyl-3,4– H]bradykinin (108 Ci mmol)1), [3,4-prolyl-3,4-3H]desArg10kallidin (80 CiỈmmol)1) and myo-[2-3H]inositol (21 CiỈ mmol)1) were from PerkinElmer Life Science (Boston, MA, USA) J F Hess (Merck, West Point, PA, USA) kindly provided us with a vector harboring the sequence of the human B1wt The antibody AS346 [6] was a generous gift from W Muller-Esterl (University of Frankfurt, ă Germany) Unlabeled peptides were bought from Bachem (Heidelberg, Germany) The primers were synthesized by Invitrogen and delivered desalted and lyophilized Pfu DNA polymerase was obtained from Stratagene Europe (Heidelberg, Germany) Fetal bovine serum, culture media, and penicillin ⁄ streptomycin were purchased from PAA Laboratories (Colbe, Germany) Fugene was from Roche ă (Mannheim, Germany) and Invitrogen supplied hygromycin B and blasticidin Poly(lysine), captopril, 1.10-phenanthroline and bacitracin were purchased from Aldrich (Taufkirchen, Germany) Ion exchange columns AG · (formiate form) were bought from Bio-Rad (Munich, Germany) All other reagents were of analytical grade and are commercially available Cell culture HEK 293 cells, host cells harboring an Flp recombinant target (FRT) site in their genome, were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, 10% (v ⁄ v) fetal bovine serum and 100 mL)1 ⁄ 100 lgỈmL)1 penicillin ⁄ streptomycin For binding studies or the measurement of total inositol phosphate accumulation cells were seeded on cell culture dishes pretreated with 0.01% (w ⁄ v) poly(lysine) in NaCl ⁄ Pi (phosphate buffered saline, PBS) to enhance their adherence Expression vectors The sequence of the human B2wt starting with the third encoded methionine [9], the sequence of the human B1wt, truncations and chimeras of both were cloned into the BamHI and the XhoI sites of the pcDNA5 ⁄ FRT vector from Invitrogen Each receptor sequence was preceded at the N-terminus by either a single hemagglutinin-tag (MGYPYDVPDYAGSA) or a double-tag (MGRSHHHHHH-GYPYDVPDYAGSA) cloned into the HindIII and BamHI site of the vector For comparison of analog positions in both receptors we used the FEBS Journal 272 (2005) 129–140 ª 2004 FEBS Role of helix and C-termini in bradykinin receptors numbering scheme of Ballesteros & Weinstein [35], where the most conserved residue in a transmembrane segment is given the number of the helix followed by the number 50 Residues proximal to this reference residue are obtained by counting down, those distal by counting up from 50 The highest conserved residue in helix 7, the proline within the NPXXY motif, is therefore named P7.50 and the tyrosine of this sequence is identified as Y7.53 Construction of mutated B1wt, B2wt and of the B1 ⁄ B2 receptor chimera Standard PCR techniques using either receptor-specific or chimeric primers with the B1wt and B2wt genes as templates were applied to generate truncated or point-mutated versions of the B1wt, B2wt and several B1 ⁄ B2 chimeras All PCR products were ligated between the BamHI and XhoI sites of the pcDNA5 ⁄ FRT vector Cells were transfected using Fugene following the manufacturer’s instructions, i.e lg plasmids (0.4 lg gene of interest in pcDNA5 ⁄ FRT plus 1.6 lg pOG44-vector) and lL Fugene per six-well dish Stably transfected clones were obtained after selection with 250 lgỈmL)1 hygromycin B [3H]Agonist binding studies For the determination of dissociation constant Kd and receptor number Bmax, confluent monolayers on 24-well plates (B2wt) ⁄ 12-well plates (B1wt) were washed three times with ice-cold PBS and incubated on ice with 0.15 or 0.3 mL of ice-cold incubation buffer [40 mm Pipes, 109 mm NaCl, mm KCl, 0.1% (v ⁄ v) glucose, 0.05% (v ⁄ v) BSA, mm CaCl2, pH 7.4; degradation inhibitors for B2wt: mm bacitracin, 0.8 mm 1.10-phenanthroline and 100 lm captopril; degradation inhibitors for B1wt: 0.5 mm bacitracin, 0.02 mm 1.10-phenanthroline and 100 lm captopril] containing increasing concentrations of [3H]BK (10 concentrations ranging from 0.01 to  40 nm) or [3H]DAK (0.01–10 nm) for at least 90 The incubation was stopped by rinsing the monolayers three times with ice-cold PBS and lysing the monolayers by addition of 0.2 mL of 0.3 m NaOH The bound radioactivity was transferred quantitatively into scintillation vials with another 0.2 mL of water and measured in a b-counter after addition of scintillation fluid Nonspecific binding was determined in the presence of lm unlabeled agonist and subtracted from the total binding to calculate the specific binding Internalization of [3H]BK and [3H]DAK To determine the internalization of receptor-bound agonist, cell monolayers on 12-well plates were rinsed three times with ice-cold PBS (pH 7.2) and incubated with the indi- 137 Role of helix and C-termini in bradykinin receptors cated concentration of [3H]agonist in 0.3 mL incubation buffer on ice to reach equilibrium binding To start the internalization of [3H]agonist the plates were transferred to a water bath at 37 °C The internalization process was stopped by placing the trays on ice at the indicated times Cells were washed three times with PBS and the remaining surface-bound [3H]agonist was removed by treating the cell monolayer with 0.2 mL of an ice-cold dissociation solution (0.2 m acetic acid ⁄ 0.5 m NaCl, pH 2.7) for 10 The dissociation solution containing the surface-bound [3H]agonist was quantitatively transferred into scintillation vials by rinsing the cell monolayer with another 0.2 mL of PBS The remaining internalized [3H]agonist was subsequently transferred to scintillation vials by lysing the cells with 0.2 mL of 0.3 m NaOH and rinsing the wells with additional 0.2 mL of water The radioactivity of both samples was determined in a b-counter after addition of scintillation fluid Nonreceptor-mediated [3H]agonist internalization was determined in the presence of lm unlabeled agonist and subtracted from the total binding to obtain the specific values Stimulation of total IP release Cell monolayers (80% confluent) in 12-well dishes were labeled for 18–24 h with 0.5 lCi of myo-[3H]inositol in 0.5 mL DMEM with fetal bovine serum and 100 mL)1 ⁄ 100 lgỈ mL)1 penicillin ⁄ streptomycin The monolayers were then placed on ice, rinsed three times with ice-cold PBS (pH 7.2) and incubated with or without the appropriate agonist in incubation buffer containing 50 mm LiCl Basal and stimulated IP accumulation was started by placing the tray in a water bath at 37 °C for 30 It was stopped by exchanging the buffer with 0.75 mL of ice-cold 20 mm formic acid and by transferring the tray onto ice for additional 30 As a baseline control one tray was left on ice with LiCl incubation buffer without agonist The EC50 was determined by adding escalating concentrations of agonist (10)12 to 10)6 m) for 30 at 37 °C The supernatant was then applied together with another 0.75 mL of 20 mm formic acid and 0.2 mL of a 3% (w ⁄ v) ammonium hydroxide solution to AG 1-X8 anion exchange columns, followed by mL 1.8% (w ⁄ v) ammonium hydroxide solution, mL of 60 mm sodium formiate, mm sodium tetraborate buffer and 0.5 mL of m ammonium formate ⁄ 0.2 m formic acid solution The total inositol phosphates were eluted by addition of 2.5 mL of the latter solution The radioactivity was determined in a b-counter after the addition of scintillation fluid All data (basal and stimulated) at 37 °C are given in fold of the amount of total IP determined in the baseline control on ice Immunoprecipitation and Western blotting Cells were washed once with PBS and solubilized in RIPA buffer [50 mm Tris ⁄ HCl, 150 mm NaCl, 1% (v ⁄ v) NP-40, 138 A Faussner et al 0.5% (w ⁄ v) sodium deoxycholate, 0.1% (w ⁄ v) SDS, mm EDTA, pH 7.5] supplemented with 0.5 mm Pefabloc SC and 10 lm each of 1.10-phenanthroline, aprotinin, leupeptin and pepstatin A for 45 at °C with gentle rocking The sample was centrifuged at 6240 g for 20 at °C and the supernatant (0.5 mL with  1.5 mg of total protein) incubated with 35 lL protein G ⁄ agarose and 2.5 lL of antiserum AS346 for h at °C The mixture was then washed twice with RIPA buffer and once with distilled water, resuspended in 30 lL of Laemmli buffer and incubated for at 95 °C After separation by electrophoresis on 10% (w ⁄ v) SDS polyacrylamide gels, the proteins were transferred onto 0.45 lm nitrocellulose membranes After blocking the membranes overnight with blocking buffer [0.25% (w ⁄ v) gelatin in 50 mm Tris ⁄ HCl, 150 mm NaCl, mm EDTA, 0.05% (v ⁄ v) Triton X-100, pH 7.5] primary high affinity anti-HA Ig (0.1 lgỈmL)1) was added in fresh blocking buffer for h at room temperature The membranes were washed twice for 10 in Tris-buffered saline with 0.1% (v ⁄ v) Tween 20 (TBST) followed by addition of the corresponding secondary peroxidase-labeled rabbit anti-rat Ig (1 : 1000) for h After washing in TBST three times each for 15 antibody binding was detected using the Western Blot Chemoluminescence Reagent Plus Receptor phosphorylation Confluent cells on 6-well plates were washed twice with phosphate-free DMEM, incubated for h at 37 °C in the same medium, and labeled with 0.2 mCiỈmL)1 [32P]orthophosphate for 10–12 h After exposure to lm BK or DAK for at 37 °C, monolayers were scraped into 0.5 mL of RIPA buffer containing protease inhibitors (see above) and phosphatase inhibitors (25 mm NaF, mm sodium orthovanadate, 0.3 lm okadaic acid) Immunoprecipitation and separation on a 10% (w ⁄ v) SDS polyacrylamide gel were carried out as described previously The proteins of interest were electroblotted onto nitrocellulose membranes and identified by autoradiography Protein determination Total protein per well was quantified by lysing the cells with 0.3 mL of 0.3 m NaOH The protein content of this solution was determined with the Micro BCA Protein assay reagent from Pierce (Rockford, IL, USA) using bovine serum albumin as standard Data analysis All data analysis was performed using graphpad prism for Macintosh, Version 3.0a (GraphPad Software, Inc., San Diego, CA, USA) FEBS Journal 272 (2005) 129–140 ª 2004 FEBS A Faussner et al Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft to A Faussner (FA 288 ⁄ 3–1) References Gether U (2000) Uncovering molecular mechanisms involved in activation of G protein-coupled receptors Endocrine Rev 21, 90–113 Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signalling Pharmacol Rev 53, 1–24 Austin CE, Faussner A, Robinson HE, Chakravarty S, Kyle DJ, Bathon JM & Proud D (1997) Stable expression of the human kinin B1 receptor in Chinese hamster ovary cells Characterization of ligand binding and effector pathways J Biol Chem 272, 11420–11425 Sabourin T, Bastian L, Bachvarov DR & Marceau F (2002) Agonist-induced translocation of the kinin B1 receptor to caveolae-related rafts Mol Pharmacol 61, 546–553 Lamb ME, Zhang C, Shea T, Kyle DJ & LeebLundberg LMF (2002) Human B1 and B2 bradykinin receptors and their agonists target caveolae-related lipid rafts to different degrees in HEK 293 cells Biochem 41, 14340–14347 Blaukat A, Herzer K, Schroeder G, Bachmann M, Nash N & Muller-Esterl W (1999) Overexpression and funcă tional characterization of kinin receptors reveal subtypespecic phosphorylation Biochem 38, 1300–1309 Pizard A, Blaukat A, Muller-Esterl W, Alhenc-Gelas F ă & Rajerison RM (1999) Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail J Biol Chem 274, 12738–12747 Ostrom RS (2002) New determinants of receptoreffector coupling: trafficking and compartmentation in membrane microdomains Mol Pharmacol 61, 473–476 Hess JF, Borkowski JA, Young GS, Strader CD & Ransom RW (1992) Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor Biochem Biophys Res Commun 184, 260–268 10 Menke JG, Borkowski JA, Bierillo KK, MacNeil T, Derrick AW, Schneck KA, Ransom RW, Strader CD, Linemeyer DL & Hess JF (1994) Expression cloning of a human B1 bradykinin receptor J Biol Chem 269, 21583–21586 11 Gutowski S, Smrcka A, Nowak L, Wu DG, Simon M & Sternweis PC (1991) Antibodies to the alpha q subfamily of guanine nucleotide-binding regulatory protein alpha subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones J Biol Chem 266, 20519–20524 FEBS Journal 272 (2005) 129–140 ª 2004 FEBS Role of helix and C-termini in bradykinin receptors 12 Marceau, F., Hess, J.F & Bachvarov, D.R (1998) The B1 receptors for kinins Pharmacol Rev 50, 358–386 13 Proud D & Kaplan AP (1988) Kinin formation: mechanism and role in inflammatory disorders Annu Rev Immunol 6, 49–83 14 Farmer SG & Burch RM (1992) Biochemical and molecular pharmacology of kinin receptors Annu Rev Pharmacol Toxicol 32, 511–536 15 Prado GN, Taylor L & Polgar P (1997) Effects of intracellular tryosine residue mutation and carboxyl terminus truncation on signal transduction and internalization of the rat bradykinin B2 receptor J Biol Chem 272, 14638– 14642 16 Faussner A, Proud D, Towns M & Bathon JM (1998) Influence of the cytosolic carboxyl termini of human B1 and B2 kinin receptors on receptor sequestration, ligand internalization, and signal transduction J Biol Chem 273, 2617–2623 17 Blaukat A, Pizard A, Breit A, Wernstedt C, AlhencGelas F, Muller-Esterl W & Dikic I (2001) Determinaă tion of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function J Biol Chem 276, 40431–40440 18 Hunyady L, Bor M, Balla T & Cat KJ (1994) Identification of a cytoplasmic Ser-Thr-Leu motif that determines agonist-induced internalization of the AT1 angiotensin receptor J Biol Chem 269, 31378–31382 19 Faussner A, Bauer A, Kalatskaya I, Jochum J & Fritz H (2003) Expression levels strongly affect ligandinduced sequestration of B2 bradykinin receptors in transfected cells Am J Physiol Heart Circ Physiol 284, H1892–H1898 20 Faussner A, Schussler S, Seidl C & Jochum M (2004) ¨ Inhibition of B2 Bradykinin receptor sequestration by phenylarsine oxide or sucrose allows determination of receptor affinity shift and ligand dissociation in intact cells Biol Chem 385, 835–843 21 Marchese A, Chen C, Kim YM & Benovic JL (2003) The ins and outs of G protein-coupled receptor trafficking Trends Biochem Sci 28, 369–376 22 Kalatskaya I, Schussler S, Blaukat A, Muller-Esterl W, ă ă Jochum M, Proud D & Faussner A (2004) Mutation of tyrosine in conserved NPXXY-sequence leads to constitutive phosphorylation and internalization, but not signaling of the human B2 bradykinin receptor J Biol Chem 279, 31268–31276 23 Perry SJ & Lefkowitz, R.J (2002) Arresting developments in heptahelical receptor signaling and regulation Trends Cell Biol 12, 130–138 24 Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, et al (2000) Crystal structure of rhodopsin: a G protein-coupled receptor Science 289, 739–745 139 Role of helix and C-termini in bradykinin receptors 25 Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices J Mol Biol 292, 195–202 26 Kang DS & Leeb-Lundberg LMF (2002) Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to Gq ⁄ 11mediated phospholipase Cb activity Mol Pharmacol 62, 281–288 27 Fathy DB, Leeb T, Mathis SA & Leeb-Lundberg LMF (1999) Spontaneous human B2 bradykinin receptor activity determines the action of partial agonists as agonists or inverse agonists J Biol Chem 274, 29603–29606 28 Ernst OP, Meyer CK, Marin EP, Henklein P, Fu WY, Sakmar TP & Hofmann KP (2000) Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits J Biol Chem 275, 1937–1943 29 Marin EP, Krishna AG, Zvyaga TA, Isele J, Siebert F & Sakmar TP (2000) The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin–transducin interaction J Biol Chem 275, 1930–1936 30 Sano T, Ohyama K, Yamano Y, Nakagomi Y, Nakazawa S, Kikyo M, Shirai H, Blank JS, Exton JH 140 A Faussner et al 31 32 33 34 35 & Inagami T (1997) A domain for G protein coupling in carboxyl-terminal tail of rat angiotensin II receptor type 1A J Biol Chem 272, 23631–23636 ` Franzoni L, Nicastro G, Pertinhez TA, Tato M, Nakaie CR, Paiva ACM, Schreier S & Spisni A (1997) Structure of the C-terminal fragment 300–320 of the rat angiotensin II AT1a receptor and its relevance with respect to G-protein coupling J Biol Chem 272, 9734–9741 Yeagle PL, Choi G & Albert AD (2001) Studies on the structure of the G-protein-coupled receptor rhodopsin including the putative G-protein binding site in unactivated and activated forms Biochem 40, 11932– 11937 Krishna AG, Menon ST, Terry TJ & Sakmar TP (2002) Evidence that helix of rhodopsin acts as a membranedependent conformational switch Biochem 41, 8298– 8309 Guex N & Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling Electrophoresis 18, 2714–2723 Ballesteros JA & Weinstein H (1995) Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein coupled receptors Methods Neursci 25, 366–428 FEBS Journal 272 (2005) 129–140 ª 2004 FEBS ... in Role of helix and C-termini in bradykinin receptors receptor signaling because: (a) interruption of the amphipathic structure of B1wt helix in B1wt and B1KB2 through the presence of a serine... TSIfiAAA N3 38* B1wt B1RB2 B1NB2 B1CB2 B1KB2 B1KB2 ⁄ SfiV B1KB2 ⁄ QGVfiKQ B1KB2 ⁄ VCfiCV B1YB2 B1V323S 10400 5020 52 98 383 2 625 127 511 1701 182 3 17 58 2142 1 786 2957 84 6 3.91 ± 1.06 3.76 ± 1.61 2 .82 ± 0.92... Faussner et al Role of helix and C-termini in bradykinin receptors Fig Schematic representation of the C-terminal B1wt and B2wt sequences and chimera thereof The C-terminal sequences beginning at transmembrane