Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes ␤-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a ␤-arrestin-independent but dynamindependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.Extracellular calcium homeostasis in vertebrate animals is primarily under the endocrine control of the parathyroid hormone (PTH) 1 /type I PTH receptor (PTH1R). The PTH1R, predominantly expressed in kidney and bone cells, belongs to class B of the large superfamily of G protein-coupled receptors (GPCRs) that consists of receptors for peptide hormones and neuropeptides (1). Class B GPCRs are characterized by a common topology and by their ability to couple to multiple signaling pathways via distinct G proteins.PTH is synthesized by the parathyroid glands as a mature peptide of 84 amino acids that is stored in secretory vesicles and dense core granules. Reductions of extracellular calcium levels are detected by the calcium-sensing receptor on parathyroid chief cells and promote the release of PTH, which acts on bone (to increase resorption) and kidney (to augment reabsorption), thereby restoring serum calcium levels. PTH-(1-84) is usually the major form of PTH secreted by the parathyroid glands. However, recent analyses reveal that PTH fragments that are likely to be PTH-(7-84) are also secreted by the parathyroid glands and generated by peripheral metabolism (2, 3). These PTH fragments or their synthetic a...

The glucagon-like peptide 1 receptor (GLP-1R) mediates important effects on beta-cell function and glucose homeostasis and is one of the most promising therapeutic targets for type 2, and possibly type 1, diabetes. Yet, little is known regarding the molecular and cellular mechanisms that regulate its function. Therefore, we examined the cellular trafficking of the GLP-1R and the relation between receptor localization and signaling activity. In resting human embryonic kidney 293 and insulinoma MIN6 cells, a fully functional green fluorescent protein-tagged GLP-1R was localized both at the cell membrane and in highly mobile intracellular compartments. Real-time confocal fluorescence microscopy allowed direct visualization of constitutive cycling of the receptor. Overexpression of K44A-dynamin increased the number of functional receptors at the cell membrane. Immunoprecipitation, sucrose sedimentation, and microscopy observations demonstrated that the GLP-1R localizes in lipid rafts and interacts with caveolin-1. This interaction is necessary for membrane localization of the GLP-1R, because overexpression of a dominant-negative form of caveolin-1 (P132L-cav1) or specific mutations within the putative GLP-1R's caveolin-1 binding domain completely inhibited GLP-1 binding and activity. Upon agonist stimulation, the GLP-1R underwent rapid and extensive endocytosis independently from arrestins but in association with caveolin-1. Finally, GLP-1R-stimulated activation of ERK1/2, which involves transactivation of epidermal growth factor receptors, required lipid raft integrity. In summary, the interaction of the GLP-1R with caveolin-1 regulates subcellular localization, trafficking, and signaling activity. This study provides further evidence of the key role of accessory proteins in specifying the cellular behavior of G protein-coupled receptors.

We previously characterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as the clinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine (ZOX), as pharmacological activators of the intermediate-conductance Ca(2+)-activated K(+) channel, hIK1. The mechanism of activation of hIK1, as well as the highly homologous small-conductance, Ca(2+)-dependent K(+) channel, rSK2, was determined following heterologous expression in Xenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced a concentration-dependent activation of hIK1, with half-maximal (K(1/2)) values of 84 microM and 98 microM, respectively. Similarly, CZ activated rSK2 with a K(1/2) of 87 microM. In the absence of CZ, the Ca(2+)-dependent activation of hIK1 was best fit with a K(1/2) of 700 nM and a Hill coefficient (n) of 2.0. rSK2 was activated by Ca(2+) with a K(1/2) of 700 nM and an n of 2.5. Addition of CZ had no effect on either the K(1/2) or n for Ca(2+)-dependent activation of either hIK1 or rSK2. Rather, CZ increased channel activity at all Ca(2+) concentrations (V(max)). Event-duration analysis revealed hIK1 was minimally described by two open and three closed times. Activation by 1-EBIO had no effect on tau(o1), tau(o2), or tau(c1), whereas tau(c2) and tau(c3) were reduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. In conclusion, we define 1-EBIO, CZ, and ZOX as the first known activators of hIK1 and rSK2. Openers of IK and SK channels may be therapeutically beneficial in cystic fibrosis and vascular diseases.

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of nonfunctional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu 406 (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27°C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca 2؉ -dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca 2؉ -dependent K ؉ channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His 299 -Lys 427 . Furthermore, truncation of hIK1 at Leu 414 resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/ rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg 355 -Ala 413 . An alanine scan of all nonconserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg 355 -Met 368 of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was ϳ50% of that of hIK1. These results demonstrate that amino acids Arg 355 -Ala 413 within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATPdependent phosphorylation of hIK1 or an associated protein is independent of Ca 2؉ .The human intermediate conductance K Ca channel, hIK1, is required for a variety of physiological processes including transepithelial ion transport (2-5), vasodilation (6, 7), T cell activation (8, 9), cell proliferation (9 -11), and regulatory volume decrease (9, 10). In addition to demonstrating modulation by intracellular Ca 2ϩ , we and others have demonstrated that hIK1 activity can be dynamically regulated by phosphorylation (1,9,(12)(13)(14). Several of these studies have demonstrated an ATP-dependent activation of hIK1 in excised, inside-out membrane patches that can be reversed by exogenous phosphatases and/or kinase inhibitors (1,12,13). Based on these observations, we speculated that the phosphorylation-dependent modulation of hIK1 plays a critical role in modulating the physiological processes in which hIK1 is involved.In our previous study we demonstrated, in excised, insideout patches, that addition of ATP (1 mM) resulted in, on average, a 3-fold increase in hIK1 activity (1), having an EC 50 of 50 M.1 The stimulatory effect of ATP exhibited a slow onset, requiring several minutes for the maximal response, was strictly Mg 2ϩ -dependent, and could be mimicked by neither hydrolyzeable (ADP, GTP, UTP, CTP, ITP) nor non-...

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G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.

Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G sand G q -mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and ␤-arrestin1 (␤-arrestin1-(319 -418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signal- ]PTHrP-(1-36)NH 2 ), which selectively activates the G s /cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G q (but not G s ) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor. Parathyroid hormone (PTH)1 is the primary regulator of serum calcium homeostasis and bone metabolism. In response to low blood calcium levels, PTH is released into the circulation and acts primarily on the receptor for the PTH/PTH-related peptide (PTHrP) in bone and kidney, PTH1R (1). PTHrP was first described as the hormone responsible for hypercalcemia of malignancy (2-4) and has subsequently been described as having a role in both cell proliferation and differentiation (5).The identification of the PTH1R as a seven-transmembrane class II G protein-coupled receptor (GPCR) (6) was followed shortly thereafter by the elucidation of associated signaling mechanisms (7,8). Upon stimulation with either PTH or PTHrP, a G protein-mediated cascade of events occurs via G s and G q , resulting in activation of the cAMP/protein kinase A and phospholipase C/protein kinase C pathways, respectively. In turn, these events lead to receptor phosphorylation (9 -11) and ␤-arrestin recruitment coupled to receptor desensitization (12-14). Agonist-receptor complex internalization then occurs via clathrin-coated pits in a ␤-arrestin-dependent manner (12,15).Many of the actions of PTH are mediated by cAMP, including its well established skeletal anabolic action (16). We have demonstrated that regulation of cAMP production by the PTH1R is predominantly under the control of ␤-arrestins (12, 15). PTH1R activation is rapidly followed by arrestin recruitment t...