Corticotropin-Releasing Hormone Receptor (CRHR)1 and CRHR2 Are Both Trafficking and Signaling Receptors for Urocortin (original) (raw)

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1Pennington Biomedical Research Center (H.T., A.J.K., W.P.), Baton Rouge, Louisiana 70808;

2Shanghai Cancer Institute (H.T.), 200032 Shanghai, China

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1Pennington Biomedical Research Center (H.T., A.J.K., W.P.), Baton Rouge, Louisiana 70808;

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1Pennington Biomedical Research Center (H.T., A.J.K., W.P.), Baton Rouge, Louisiana 70808;

*Address all correspondence and requests for reprints to: Weihong Pan, M.D., Ph.D., Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808.

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Received:

09 December 2005

Accepted:

05 December 2006

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Hong Tu, Abba J. Kastin, Weihong Pan, Corticotropin-Releasing Hormone Receptor (CRHR)1 and CRHR2 Are Both Trafficking and Signaling Receptors for Urocortin, Molecular Endocrinology, Volume 21, Issue 3, 1 March 2007, Pages 700–711, https://doi.org/10.1210/me.2005-0503
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Abstract

Transport of urocortin, a potent satiety peptide, occurs at the blood-brain barrier of the mouse. Endocytosis of urocortin by the cerebral microvessel endothelial cells composing the blood-brain barrier is a rate-limiting step of this transport, but the cellular mechanisms involved have not been fully elucidated. The presence of both CRH receptors R1 and R2 in isolated cerebral microvessels shown in this study suggested that both subtypes might mediate urocortin transport. The roles of these two receptors in the endocytosis and signal transduction of urocortin were tested by overexpression studies in human embryonic kidney 293 cells. Both receptors led to a significant increase of binding and endocytosis of radiolabeled urocortin. CRHR1-mediated urocortin endocytosis was blocked by astressin (antagonist for both CRHRs), whereas CRHR2-mediated urocortin endocytosis was also blocked by antisauvagine 30 (selective CRHR2β antagonist). Chlorpromazine, filipin, and nystatin had no effect on urocortin endocytosis, indicating the lack of significant involvement of clathrin or caveolae membrane microdomains. Both CRHR1 and CRHR2 were able to mediate the ligand-induced increase of cAMP production, suggesting that the overexpressed receptors were biologically active. Elevation of intracellular cAMP by forskolin or dibutyryl-cAMP, however, did not show acute modulation of the binding and endocytosis of urocortin. Despite the substantial intracellular degradation of endocytosed urocortin in cells overexpressing either CRHR1 or CRHR2, intact urocortin could be exocytosed during the 1-h study interval. We conclude that both CRHR1 and CRHR2 play a facilitatory role in the non-clathrin-, non-caveolae-mediated endocytosis and intracellular signal transduction of this potent peptide.

UROCORTIN 1 IS FOUND in both the periphery and brain. Peripheral administration of urocortin decreases food intake and increases energy expenditure (1–3). Leptin potentiates this satiety effect, facilitating urocortin permeation across the blood-brain barrier (BBB) (4, 5). This blood-to-brain saturable transport system for urocortin is inhibited by an antagonist of the CRH receptor (CRHR)-2 (6), to which urocortin binds 41 times more strongly than does CRH (7).

Both urocortin and CRH bind to their cognate receptors, CRHR1 and CRHR2, to activate G protein-coupled signal transduction pathways and cAMP production. Because of the selectively high binding of urocortin to CRHR2, much attention has been focused on their relationship, including the apparently greater role of CRHR2 than CRHR1 for the satiety effects of urocortin (8, 9). Yet, the binding affinity of urocortin to CRHR1 is 6-fold higher than that of CRH (7) and, in the brain, CRHR1 has a wider regional distribution than CRHR2 (10).

CRHR1 and CRHR2 are differentially distributed in the body, but results from RT-PCR in our present study showed that they are expressed equally in primary brain microvessel endothelial cells. Therefore, both receptors may be closely involved in the transport of urocortin across the BBB, particularly after activation by leptin, TNFα, or hyperglycemia (4, 11, 12). Having shown that leptin-activated urocortin transport across the BBB in the mouse is partially mediated by CRHR2, being inhibited by the CRHR2β receptor antagonist antisauvagine 30 (6), we further examined the relative roles of CRHR1 and CRHR2 in the transport of urocortin by use of cell lines overexpressing either receptor after transient transfection. We found that CRHR1, as well as CRHR2, facilitated the endocytosis of urocortin that apparently occurs by a non-clathrin-, non-caveolae-mediated pathway.

RESULTS

Both CRHR1 and CRHR2 Are Present in Cerebral Endothelial Cells

Equal amounts (40 μg) of proteins from cell or tissue lysates were electrophoresed and immunoblotted for expression of CRHR. CRHR1 has an expected molecular mass of 47.768 kDa and CRHR2 has an expected molecular mass of 49.682 kDa. The observed molecular mass of CRHRs was about 55 kDa, probably related to glycosylation of the receptors. Although CRHR protein expression was present in freshly isolated cerebral microvessels (lane 4) as well as whole brain homogenate (lane 3), and to a lesser extent in the hypothalamus (lane 1) and total pituitary (lane 5), it was many times lower in cultured TM-BBB4 cells (lane 2) (Fig. 1A). Because CRHR1 and CRHR2 have similar molecular masses and were difficult to differentiate by size in the Western blot by use of a shared antibody, RT-PCR was further performed in freshly isolated mouse cerebral microvessels. The result was further verified by nested PCR. The mRNA for CRHR1 and CRHR2 had similar levels of expression (Fig. 1B). Because cultured TM-BBB4 endothelial cells did not have abundant CRHR1 or CRHR2 expression, we used human embryonic kidney (HEK)293 cells (which have higher transfection efficiency than TM-BBB4 cells) to further test the roles of receptor subtypes in the endocytosis of urocortin by overexpression studies. The molecular masses of overexpressed CRHR1 and CRHR2 were both about 55 kDa. To further determine the differential protein expression of the receptor subtypes, two subtype-specific antibodies were used for immunofluorescent staining and Western blot analysis. By immunofluorescent staining of HEK293 cells overexpressing either CRHR1 or CRHR2 receptors, we show in Fig. 1C that there was no cross-reactivity of the CRHR1 and CRHR2 antibodies. Each of the receptor subtypes showed punctated cytoplasmic staining, including predominant perinuclear distribution in some cells. Furthermore, Fig. 1D shows that both CRHR1 and CRHR2 contributed to the strong signal detected in the cerebral microvessel preparation.

Fig. 1.

Expression of CRHRs in Mouse Brain and Cellular Models A, Protein expression of CRHRs was shown by Western blot in the mouse hypothalamus, total brain homogenate, cerebral microvessel preparation, and total pituitary. A specific signal at about 55 kDa was detected. By contrast, expression in the cultured mouse cerebral endothelial cell line TM-BBB4 was below the limits of detection. B, mRNA for both CRHR1 and CRHR2 was present in the cerebral microvessel preparation of mice as shown by RT-PCR. C, Immunofluorescent staining of HEK293 cells overexpressing either CRHR1 or CRHR2 showed that the subtype-specific antibodies did not have cross-reactivity. D, The presence of CRHR1 and CRHR2 signals in cerebral microvessels was shown by Western blot analysis. MW, Molecular mass; kD, kilodalton.

Both CRHR1 and R2 Mediate Binding and Internalization of Urocortin

The surface binding and endocytosis of [125I]urocortin at 30 min were significantly (P < 0.005) increased in HEK293 cells 24 h after transfection with 2 μg/well of either CRHR1 or CRHR2 DNA (Fig. 2A). The groups overexpressing CRHR1 and CRHR2 showed no significant differences in [125I]urocortin uptake when compared with each other.

Fig. 2.

Effect of Receptor Overexpression and Antagonists on Urocortin Uptake A, Binding and endocytosis of [125I]urocortin were significantly increased in HEK293 cells by overexpression of either CRHR1 or CRHR2. The cells were incubated with [125I]urocortin for 30 min at 37 C. There were no significant differences between the CRHR1 and CRHR2 groups. ***, P < 0.005 compared with the vector control. B, Endocytosis of [125I]urocortin was studied in HEK293 cells 50 h after transient transfection. The nonselective antagonist to CRHR1 and CRHR2 had a significant inhibitory effect in the endocytosis (P < 0.05), whereas the CHRR2-selective antagonist antisauvagine 30 did not cause a significant change. C, Significant inhibitory effects of receptor antagonists on binding and endocytosis of [125I]urocortin were shown in cells overexpressing CRHR2 receptors when the CRHR2-overexpressing group was compared with either the vector-only control or CRHR2 overexpression in the presence of antisauvagine 30, astressin, or αhCRH along with [125I]urocortin. ***, P < 0.005.

To provide further evidence of the specific effects of CRHRs in mediating the uptake of [125I]urocortin, CRHR receptor antagonists were used. In HEK293 cells 50 h after transient transfection of CRHR1, the nonselective receptor antagonist astressin significantly inhibited the endocytosis of urocortin, whereas the CRHR2β antagonist antisauvagine 30 had no effect (Fig. 2B). The higher percentage of endocytosis in this study indicated that more CRHR1 was expressed at 50 h than at 24 h after transfection. In cells 24 h after transient transfection of CRHR2, the uptake of urocortin was significantly decreased by the specific inhibitor antisauvagine 30 as well as by the nonselective inhibitors astressin and α-helical CRH (αhCRH) that block both CRHR1 and CRHR2 (Fig. 2C).

Endocytosis of Urocortin Does Not Involve Common Membrane Microdomains

The most common mechanisms of receptor-mediated endocytosis are clathrin- or caveolae-mediated pathways. Effective doses of chlorpromazine and filipin, known to inhibit clathrin- and caveolae-mediated endocytosis, respectively, were used in the study of urocortin binding and uptake. There was no inhibitory effect of chlorpromazine or filipin on basal urocortin uptake in native HEK293 cells. In cells overexpressing CRHR1, neither inhibitor caused a significant decrease in the surface binding or endocytosis of [125I]urocortin. The sterol-binding agent nystatin, which is known to inhibit caveolae-mediated endocytosis, caused a paradoxical increase in surface binding and endocytosis of urocortin, suggesting nonspecific membrane changes. The solvent methanol used to dissolve filipin had no significant effect on the results (Fig. 3A). Similarly, neither filipin nor chlorpromazine caused a significant decrease in the endocytosis of urocortin in CRHR2-overexpressing cells (Fig. 3B). These results contrast with the significant inhibitory effects of chlorpromazine, filipin, and nystatin on the internalization of [125I]TNFα (Fig. 3C).

Fig. 3.

Effect of Inhibitors of Membrane Microdomains on Ligand Endocytosis A, In cells overexpressing CRHR1, there was no significant inhibitory effect of chlorpromazine (against clathrin coat assembly), filipin and nystatin (both disrupt caveolae), or the solvent methanol on binding and internalization of [125I]urocortin. B, In cells overexpressing CRHR2, chlorpromazine and filipin also did not show a significant inhibitory effect. C, In TM-BBB4 cells, filipin, chlorpromazine, and nystatin caused a significant decrease in the endocytosis of [125I]TNFα. By contrast, methanol and xylazine, which were used to dissolve the compounds, had no significant effect. *, P < 0.05; **, P < 0.01.

Overexpressed Receptors Mediate Increased cAMP Production after Urocortin Stimulation

In the absence of CRHR1 or CRHR2 receptors, the baseline production of cAMP in HEK293 cells was low, despite stimulation by urocortin for 10 min. In the absence of urocortin stimulation, there was no significant cAMP production even though the cells overexpressed CRHR1. When urocortin was added to the cells overexpressing either CRHR1 or CHRR2, both groups showed more than a 4-fold increase of cAMP production, indicating ligand-dependent signal transduction mediated by both receptors (Fig. 4A). Futhermore, forskolin and db-cAMP did not affect urocortin binding and endocytosis (Fig. 4B).

Fig. 4.

Effect of Intracellular cAMP on Urocortin Uptake A, Intracellular signal transduction mediated by CRHR1 or CRHR2 was determined by cAMP assays. Receptor overexpression alone or urocortin alone in HEK293 cells transfected with the control pcDNA3.1 plasmid, did not cause significant cAMP production. Stimulation of the cells overexpressing either CRHR1 or CHRR2 by urocortin led to a significant increase in cAMP production. ***, P < 0.005. B, In HEK293 cells overexpressing CRHR1 or CRHR2, there was a significant increase in both binding and endocytosis of [125I]urocortin after 20 min of incubation compared with the mock-transfected cells. Neither forskolin nor db-cAMP caused a significant increase of the binding and endocytosis in any group. UCN, Urocortin.

Receptor Overexpression Increases the Amount of Urocortin Exocytosed but Also Increases Its Degradation

In assays for receptor exocytosis and recycling of transferrin, incubation of the cells in pH 5 buffer followed by washes with efflux medium promoted ligand-receptor dissociation at the cell surface (15, 16). To ensure that the pH 5 wash was effective in the measurement of urocortin exocytosis, control experiments were performed (Fig. 5A). The protein-denaturing enzyme trypsin reduced the amount of exocytosis, although the treatment caused detachment of some cells. Trypsinization after pH 5 wash did not cause a significant decrease in the intracellular pool of urocortin (Fig. 5A, inset). Endocytosis in the presence of αhCRH or at 4 C caused a significant decrease in both the percentage of urocortin exocytosed and that remaining inside the cells at the end of the study. The dynamic increase of urocortin recovered from the exocytosis medium over time further supports the effectiveness of the assay.

Fig. 5.

Exocytosis of Urocortin after Internalization A, A control experiment for exocytosis showed that the amount of urocortin exocytosed was significantly lower after trypsin treatment, αhCRH, or at 4 C. The percentage of urocortin remaining inside the cells at the end of the 30 min exocytosis also showed a significant decrease in the 4 C endocytosis group, a trend (+, P = 0.08) toward a decrease by αhCRH, but no significant change by trypsin (inset). **, P < 0.01; ***, P < 0.005. B, Efflux of [125I]urocortin was linear between 2.5 and 60 min after the cells were allowed to internalize [125I]urocortin for 30 min. Overexpression of CRHR1 or CRHR2 caused a significant increase in the efflux rate of radiotracer (P < 0.005 compared with the vector control). C, CRHR1 and CRHR2 transfected groups had significantly (P < 0.005) more degradation of internalized [125I]urocortin shown by acid precipitation of the radiotracer in the efflux medium.

To test the time course of exocytosis after the cells were loaded with [125I]urocortin for 30 min, cells were incubated in fresh medium for 2.5–60 min, with different triplicated wells of cells representing each time point. The percent radioactivity released into the efflux medium showed a linear regression with time in each of the three groups. The vector control had an efflux rate of 0.138 ± 0.050 (%/min), whereas the cells overexpressing CRHR1 and CRHR2 had efflux rates of 0.413 ± 0.036 and 0.413 ± 0.037, respectively. The increase from the control group was significant for both the CRHR1 and CRHR2 groups (P < 0.005). By the end of the study (60 min), about 26% of the radioactivity was exocytosed in cells overexpressing the receptors, by contrast with the 17% exocytosed in pcDNA3.1 vector-transfected cells (Fig. 5B). Although the radioactivity exocytosed in the control group mainly represented intact [125I]urocortin, as shown by acid precipitation, less than half of the total amount of radioactivity in the medium of cells overexpressing CRHR1 or CRHR2 remained as intact [125I]urocortin. The CRHR2 overexpressing cells showed less degradation of [125I]urocortin than the CRHR1 overexpressing cells (P < 0.005) when the results were analyzed by two-way ANOVA (Fig. 5C). Overall, overexpression of either receptor was associated with significantly more degradation of the endocytosed [125I]urocortin.

The efficiency of urocortin cycling through to the cell culture medium was calculated as follows: The CRHR1- or CRHR2-transfected cells had 26% exocytosis of the internalized [125I]urocortin at 60 min, 30% of that being acid precipitable, suggesting that 7.8% of the internalized urocortin exited the cells in intact form. The control group transfected with vector only exocytosed 17%, but 90% of that was acid precipitable at 60 min, with 15.3% of the internalized peptide being released in intact form. Because receptor overexpression led to more than a 4-fold increase in the [125I]urocortin internalized at 30 min, as shown in Fig. 2A, receptor overexpression showed an overall increase of at least 2-fold in the amount of intact [125I]urocortin exocytosed. Thus, overexpression of CRHRs in the cells facilitated endocytosis as well as expedited intracellular degradation, but the net effect was increased exocytosis.

Further Analysis of Degradation Patterns by Gel Autoradiography and Size-Exclusion Chromatography

Cell lysates collected 20 min after endocytosis of [125I]urocortin were electrophoresed along with stock solutions of [125I]urocortin and free 125I. Most of the signal was detected at the position of 125I-urocortin, indicating that endocytosed urocortin remained intact (Fig. 6A). To further estimate the percentage of degradation, cell lysates and the exocytosis medium were analyzed by P4 size exclusion chromatography. Figure 6B shows that all the radioactivity eluted at the position of intact [125I]urocortin 20 min after endocytosis. This pattern persisted even 1 h after endocytosis. However, exocytosed peptide was mainly degraded after endocytosis by cells transfected with either CRHR1 or CRHR2, by contrast with the limited degradation in cells transfected with empty vector only (Fig. 6C).

Fig. 6.

Stability of Endocytosed Urocortin A, Gel autoradiography of cell lysates obtained after mock transfection or CRHR1 /CRHR2 overexpression. In all groups, the signal was present at 4.7 kDa, the same as in the stock solution. No band of lower molecular mass was seen, indicating that the biological samples did not contain degraded [125I]urocortin. B, Endocytosed [125I]urocortin remained intact in groups of cells that overexpressed CRHR1, CRHR2, or neither receptor. C, Exocytosed [125I]urocortin had substantial degradation in cells overexpressing CRHR1 or CRHR2. kD, Kilodalton.

DISCUSSION

Urocortin is produced in both peripheral tissue and the central nervous system (CNS). The CRHR protein also is present in the endothelial cells composing the BBB (17). In microvessel-enriched fractions from cerebral cortex, the mRNA and protein for CRHR1 and CRHR2 showed equal amounts of expression, as indicated by RT-PCR and Western blot with subtype-specific antibodies. This provides a basis to study the mechanisms of endocytosis and signal transduction of urocortin mediated by the CRHRs. To do this, we overexpressed the receptors in HEK293 cells because of the high transfection efficiency of this cell line.

The plasma level of the 4.7-kDa urocortin measured by RIA is about 12–17 pg/ml in humans (18) and 15–19 pmol/liter in sheep (19). Because the [125I]urocortin used in our study had a specific activity of 60 Ci/g (i.e. 1 μCi = 0.017 μg), the concentration of 700,000 cpm/ml of [125I]urocortin represents 0.3 μCi/ml or 5 ng/ml. Therefore, the doses used for transport assays and cAMP stimulation were higher than physiological levels, but high doses of peripheral urocortin have been shown to exert pronounced cytoprotective effects in several studies. Urocortin decreases ischemic-reperfusion damage (10−11 to 10−8m) (20), protects against pacing-induced heart failure (0.3 μg/kg·h for 30 min in sheep) (21), reduces food intake (0.003 to 3 nmol) (1, 22), and inhibits operant responses for food reward (5–10 μg/ml/kg in rats). The concentrations of urocortin we chose fall within the range of previous studies showing circulating urocortin to be not only physiologically important but also pharmacologically beneficial in its cytoprotective effects, which are at least partially mediated by vascular endothelial cells.

There were several unusual observations in this study. First, we showed that CRHR1 played an equal role with CRHR2 in the binding and endocytosis of urocortin. After expression of equal amounts of either receptor, the amplitudes of increase in the surface binding and endocytosis of [125I]urocortin were not significantly different from each other. This broadens the perspective on the role of CRHR1 in the CNS, because most reports deal with the predominant effect of CRHR2 in mediating the actions of urocortin (8, 9). The question arises as to how CRHR1- and CRHR2-mediated uptake of urocortin by cerebral vascular endothelial cells translates into delivery of urocortin to its sites of action within the CNS. Most of the existing literature focuses on the distribution of receptors in parenchymal cells (10). CRHR1 expression is diffuse in the CNS and enriched in the cerebral cortex and cerebellum, whereas CRHR2 is more localized to subcortical structures including the limbic system and hypothalamic nuclei. Using endothelial-enriched preparations, we provide the first evidence that both CRHR1 and CRHR2 are expressed at the BBB level. The receptor subtypes play equal roles shown by overexpression and transport assays. Although there may be regional differences in differential CRHR receptor expression at the BBB level, the outcome of complete transcytosis of urocortin is best determined by the distribution of receptor subtypes on specific parenchymal cells.

A second unusual observation is that receptor-mediated endocytosis of urocortin did not seem to occur by the most common mechanisms, i.e. clathrin- or caveolae-mediated internalization, as shown by the lack of effect of chlorpromazine and filipin at doses known to be effective (23). The significant inhibitory effects of both chlorpromazine and filipin on the endocytosis of TNFα in our own studies also served as positive controls. Although HEK293 may not express abundant caveolin-1, the expression of clathrin has been consistent (24–26). Even allowing for differences in cell lines and the possible involvement of clathrin or caveolae in BBB transport of urocortin in intact animals, we can conclude that receptor-mediated endocytosis of urocortin in HEK293 cells does not involve either membrane microdomain. There are other means of pinocytosis that probably involve different intracellular mechanisms as well as membrane microdomains (27–31). It also has been shown that clathrin-mediated endocytosis involves the large GTPase dynamin and adaptor protein-2, whereas clathrin-independent endocytosis may involve flotillin-1 in lipid rafts, particularly for glycosylphosphatidylinositol-linked proteins and cholera toxin B subunit (32). Further work should address the involvement of cellular motors and raft-associated integral membrane proteins in the ligand-induced endocytosis of G protein-coupled receptors. Identification of the mechanisms of endocytosis will facilitate better understanding of how urocortin transport is related to its physiological functions.

As expected, the overexpressed CRHRs mediated not only endocytosis, but also signal transduction by production of cAMP after treatment with urocortin, indicating activation of G protein and protein kinase A by these receptors. Because we did not include the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) in lysing the cells, the amplitude of increased cAMP was probably underestimated. It is not certain how the signal transduction and intracellular trafficking of urocortin interact with each other. However, urocortin and CRH are known to modulate the permeability of the BBB in response to stress or ischemia (17), and increased intracellular levels of cAMP have been shown in cultured systems to reinforce tight junctions and reduce the permeability of the BBB (33, 34). We, therefore, further tested whether increased intracellular cAMP induced by either forskolin or db-cAMP could modulate urocortin binding and endocytosis. In neither native cells nor those overexpressing CRHRs did these reagents cause significant elevation of urocortin binding or endocytosis, indicating that there was no acute modulation.

A third novel observation concerns the stability and exocytosis of internalized urocortin. Gel autoradiography, size-exclusion chromatography, and acid precipitation all indicated that endocytosed urocortin remained intact even 1 h later. Thus, the action of urocortin persisted long after its internalization. Although most studies of peptide signaling still mainly address receptor-mediated endocytosis, with little attention to intracellular fate and ultimate transcytosis, urocortin exhibited substantial efflux in intact form in HEK293 cells in this study, both with and without receptor overexpression. The basal transport of urocortin was inhibitable by excess unlabeled urocortin, and this can be consistent with alternative mechanisms involving ion channels at urocortin binding sites on the cell surface (35), even though the receptors play a dominant role. Overexpression of CRHR1 and CHRR2 caused more than a 4-fold increase in the endocytosis of urocortin at 30 min, but the percentage of exocytosis did not show a correspondingly large increase. Rather, groups with receptor overexpression showed more intracellular degradation of urocortin; of the total radioactivity endocytosed at 30 min, acid precipitation showed that only 7.8% of the urocortin released into the cell culture medium remained intact in the overexpressed groups, by contrast with 15.3% in the control group. When analyzed by size exclusion chromatography, the extent of degradation was similar. Thus, by contrast with endocytosis, the CRHRs did not play an essential role in the exocytosis of urocortin. Regardless, there was at least a 2-fold increase in the exocytosis of intact urocortin induced by either CRHR. Therefore, despite increased intracellular degradation, this amount still represents a marked increase in exocytosis.

There are drawbacks to the use of HEK293 cells as the cellular model despite the high efficiency of transfection because they do not exhibit the polarization of the endothelial cells composing the BBB and may possess different degradative mechanisms for internalized peptides. However, endocytosis of urocortin is mainly determined by membrane microdomains and specific receptors; therefore, observations from overexpression studies should be comparable to those in cerebral microvessel endothelial cells. In cerebral endothelial cells with polarized sorting, it is possible that the exocytosis of urocortin might be even greater than that seen in HEK293 cells overexpressing CRHRs.

In summary, we showed that not only CRHR2 but also CRHR1 is involved in the endocytosis of [125I]urocortin. Although this endocytosis was decreased by antagonists to the CRHRs, it was not reduced by inhibition of the assembly of clathrin-coated pits or caveolae, indicating a nonclathrin, noncaveolae mechanism of transport. Both CRHR1 and CRHR2 mediated cAMP production after urocortin stimulation, but neither was directly involved in the exocytosis of intact [125I]urocortin. Because urocortin plays important neuromodulatory roles by crossing the BBB, information about its receptor-mediated transport and subsequent intracellular trafficking may facilitate therapeutic manipulations such as targeted drug delivery.

MATERIALS AND METHODS

HEK293 cells were obtained from American Type Culture Collection (Manassas, VA). TM-BBB4 cells were kindly provided by Dr. Tetsuya Terasaki in Sendai, Japan. Rat urocortin (Synpep, Dublin, CA) was radioactively labeled (radiolabeled) with 125I by the iodogen method and purified on a column of Sephadex G-10. The specific activity was 40–60 Ci/g. The acid precipitability of [125I]urocortin was greater than 96% for all experiments. Mouse recombinant TNFα (R&D Systems, Minneapolis, MN) was similarly radiolabeled by the iodogen method and used in control studies. The CRHR antagonists were antisauvagine 30 (4 μg/ml, Phoenix Pharmaceuticals, Belmont, CA), α-helical CRH (αhCRH; 6 μg/ml, Phoenix Pharmaceuticals), and astressin (4 μg/ml; American Peptide, Sunnyvale, CA). Chlorpromazine (25 μg/ml) was used as an inhibitor for clathrin-coated pit formation at the cell membrane. The sterol-binding agents nystatin (5 μg/ml) and filipin (5 μg/ml) were used as inhibitors for caveolae-mediated endocytosis, whereas the non-sterol-binding xylazine (5 μg/ml), which also inserts into the cell membrane, was used as a control. db-cAMP (100 μm), forskolin (25 μm), and IBMX (300 μm) were used to increase intracellular cAMP levels. Trypsin (0.05%) was used in the exocytosis control study. Methanol was used as solvent for some agents in the endocytosis assay and included as the vehicle control groups. Unless specified, all reagents were obtained from Sigma (St. Louis, MO).

Plasmid Construction

RNA was extracted from dorsal midbrain containing the Edinger-Westphal nucleus (for CRHR1) and cerebral cortex (for CRHR2) of C57 mice with an Absolutely RNA extraction kit (Stratagene, La Jolla, CA). After reverse transcription, cDNA was subjected to PCR amplification by two sets of primer specific for full-length CRHR1 and CRHR2β: CRHR1 forward (F), 5′-CGGAATTCGCCACCATGGGACAGCGCCCGCAGCTC-3′, CRHR1 reverse (R), 5′-CGGGATCCTCACACTGCTGTGGACTGCTTGAT-3′; CRHR2 F, 5′-GGGAATTCGCCACCATGGGGACCCCAGGCTCTCTTC-3′; CRHR2 R, 5′-GGGAAGCTTTCACACAGCAGCTGTCTGCTTG-3′. Full-length CRHR1 (1248 bp) and CRHR2 (1291 bp) coding sequences were successfully cloned into the pcDNA3.1(-) vector. The restriction enzyme sites for CRHR1 were _Eco_RI and _Bam_HI, and those for CRHR2 were _Eco_RI and _Hin_dIII. The plasmids were verified by restriction digestion and DNA sequencing, and the protein expression was confirmed by Western blot and immunofluorescent staining.

Transfection

Constructs of full-length CRHR1 and CRHR2 were subcloned into pcDNA3.1 and the correct insertion was verified by sequencing. HEK293 cells were grown in DMEM supplemented with 10% fetal bovine serum and antibiotics at 37 C with 5% CO2. For transient transfection with lipofectamine 2000 (Invitrogen, Carlsbad, CA) in serum-free medium, cells were grown in six- or 12-well plates overnight to achieve about 90% confluency. Equal amounts of vector-only control or CRHR1 and CRHR2 plasmid DNA (2 μg/well for six-well plates, or 0.8 μg/well for 12-well plates) were used, with triplicates of wells in each group. The cells were studied 18–50 h after transfection. The effective transfection and high level of expression were verified by Western blot analysis.

Analysis of CRHR mRNA Expression by RT-PCR

Microvessels were obtained from the cerebral cortex of adult C57 mice by capillary depletion as previously described (13). Total RNA was extracted with an Absolutely RNA extraction kit (Stratagene). Synthesis of cDNA was performed by use of the SuperScript III First-Strand Synthesis System (Invitrogen). The following primers were used for PCR amplification: CRHR1 forward, 5′-AGTGCTGGTTTGGCAAACGT-3′; reverse, 5′-CCCTGGAGACCTCGTCCT-3′; CRHR2 forward, 5′-CAGGGTTTCTTTGTGTCCGT-3′; and reverse, 5′-GTCTGCTTGATGCTGTGGAA-3′. The PCR involved 30 cycles of denaturing at 95 C for 20 sec, 55 C for 30 sec, and 68 C for 1 min. PCR product (5 μl) was electrophoresed on 1% agarose gel. The primers used are located at different exons, and the specificity of the product was further verified by nested PCR.

Analysis of CRHR Protein Expression by Western Blot and Immunofluorescent Staining

To test the level of CRHR protein expression, we used equal amounts of proteins from whole brain homogenate, hypothalamus, total pituitary, mouse cerebral microvessel preparation, and cultured TM-BBB4 mouse cerebral microvessel endothelial cells. The proteins were loaded onto 10% polyacrylamide gel and electrophoresed. The proteins were transferred to nitrocellulose membrane and probed with a primary goat antibody against the C terminus of the domains shared by both CRHR1 and CRHR2 (sc-1757; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After subsequent incubation with an horseradish peroxidase-conjugated secondary antibody, the signal was developed with an Enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL).

To determine which subtype of CRHR had a higher level of expression in cerebral microvessels, Western blot was performed on 40 μg of electrophoresed microvessel proteins freshly isolated from mouse cerebral cortex and lysed in radioimmune precipitation assay buffer containing a protease inhibitor cocktail. The membrane was probed with antibodies against CRHR1 (sc-12381), CRHR2 (sc-1826), or the shared epitope as above (sc-1757) from Santa Cruz Biotechnology. To ensure that the antibodies against CRHR1 and CRHR2 were indeed subtype specific and did not have cross-reactivity, HEK293 cells grown on coverslips were transfected with CRHR1 or CRHR2 plasmids along with an empty vector (pcDNA3.1) control. At 24 h after transfection, the cells were fixed with 3% paraformaldehyde, permeabilized with Triton X-100, and blocked with 10% normal serum. The cells were then incubated with primary antibodies against CRHR1 or CRHR2 and Alexa 488-conjugated secondary antibody, with thorough washes in between. The coverslips were mounted and fluorescent images were obtained.

Uptake of [125I]Urocortin

To test whether CRHR1 or CRHR2 overexpression led to increased binding and endocytosis, and to further test whether receptor antagonists and inhibitors for receptor-mediated or adsorptive endocytosis modulate this process, groups of cells were studied in triplicates of wells with designs specified in Results. Thirty minutes before the study, HEK293 cells were equilibrated in transport buffer (DMEM containing 25 mm HEPES and 0.5% albumin) at 37 C and pretreated for 20 min with the modulators for endocytosis as previously indicated. [125I]urocortin (700,000 cpm/ml) was added at time 0 in the presence or absence of endocytosis modulators, and the plates were placed in a 37 C shaking water bath until the end of the study (0–60 min), at which time the plates were transferred to ice and the transport buffer was rapidly replaced with ice-cold PBS. The 0 min group was maintained on ice throughout. A mild acid wash was performed with 0.2 n acetic acid, pH 3.5. After removal of specific surface binding substances by this procedure, the cells were lysed, scraped, and collected in test tubes for further degradation assays or direct measurement of radioactivity in a γ-counter.

cAMP Assay

HEK293 cells were grown in 12-well plates and transfected with 0.8 μg of pcDNA3.1, CRHR1, or CHRR2 constructs at 90% confluency (triplicate wells for each group). The cells were preincubated 29 h later in HEPES-containing transport buffer containing 300 μm IBMX and stimulated with urocortin (100 ng/ml) for 10 min in the presence of IBMX. A control group with transfected CRHR1 was incubated with PBS rather than urocortin. At the end of the study, the cells were lysed for 45 min at 37 C in 200 μl of the lysis buffer provided with the cAMP assay kit after aspiration of the media, and the supernatant was collected after centrifugation at 10,000 × g for 5 min. Measurement of cAMP concentration was performed with a chemiluminescent ELISA kit (Applied Biosystems, Bedford, MA), following the protocol developed by Chiulli et al. (14) with minor modifications. Standards or samples (60 μl/well) in duplicate were added to the 96-well capture plate and mixed with 30 μl of cAMP-alkaline phosphatase conjugate (1:100 dilution of stock solution) and 60 μl of anti-cAMP antibody. The mixture was incubated at room temperature on a plate shaker for 1 h. The plate was then washed manually six times with wash buffer. After addition of CSPD ready-to-use chemiluminescent substrate with Sapphire-II enhancer (Applied Biosystems), the plate was incubated in the dark at room temperature for 30 min on a plate shaker. The intensity of the signal was measured with a Berthold luminometer, and the concentrations of cAMP in the samples were calculated from the standard curve. Protein concentration was determined in parallel from the lysates by use of a BCA assay kit (Pierce).

Exocytosis Assays

The cells were incubated with [125I]urocortin (700,000 cpm/ ml) at 37 C for 30 min. After removal of the medium and PBS wash, the cells were incubated with pH 5 citrate buffer (150 mm NaCl and 20 mm sodium citrate in dH2O) at room temperature for 2 min, followed by three rapid washes of prewarmed (37 C) transport/efflux buffer. This treatment has been shown to be efficient in promoting the release of transferrin from its receptor at the cell surface (15, 16). Fresh transport buffer was then added, and the cells were incubated for 2.5–60 min. The cells were lysed and the radioactivity remaining intracellular was measured. The percent urocortin exocytosed was determined from the radioactivity recovered from the transport buffer divided by the total amount of radioactivity from the transport buffer, acid wash, and cell lysate.

In a separate experiment, three control groups were studied in HEK293 cells incubated with [125I]urocortin (700,000 cpm /ml) at 37 C for 30 min. One group included the nonselective CRHR antagonist αhCRH (6 μg/ml) during the course of endocytosis. A second group included two washes with 0.05% trypsin after the cells were incubated with pH 5 buffer. A third group underwent endocytosis at 4 C rather than 37 C. However, the trypsinized cells showed visible detachment, which hampered accurate interpretation of the results. The endocytosed [125I]urocortin was also measured at the end of the exocytosis assay.

Degradation Assays

To determine whether the measured radioactivity represented intact [125I]urocortin and not dissociated free 125I or smaller peptide fragments resulting from degradation, we collected the exocytosis medium or lysed the cells with radioimmune precipitation assay buffer containing a protease inhibitor cocktail at the end of the endocytosis assay. After ultracentrifugation to remove cell debris, the supernatant was subjected to acid precipitation, size-exclusion chromatography, and gel autoradiography. For acid precipitation, an equal volume (1 ml) of 30% NaCl-oversaturated trichloroacetic acid was added to the buffer. The solution was vigorously mixed, placed on ice for 15 min, and centrifuged at 3500 × g at 4 C for 15 min. The precipitate and supernatant were carefully separated and the radioactivity measured. For gel autoradiography, 60 μl of cell lysate (2000–8000 cpm) were loaded onto 18% SDS-PAGE and electrophoresed. The electrophoresis was terminated before the runoff of the dye front (bromphenol blue, 692 Da). The gel was dried and exposed to x-ray emulsion film (Kodak Scientific Imaging, Biomax MS, emulsion number 04300104) at −70 C for 10 d. In addition to the groups (lanes) of samples specified in Results, the stock solution was also tested in parallel as a positive control. For size-exclusion chromatography, Bio-Gel P4 polyacrylamide columns (Bio-Rad Laboratories, Hercules, CA) were used. The columns had bead sizes of 90–180 μm and a separation range of 800-4000 Da. The column size was 6 mm × 9 cm. Fractions (200 μl) were collected after elution of cell lysate or exocytosis medium with PBS at a flow rate of 6 min/ml.

Statistical Analyses

Means are shown with their ses. To compare the difference among groups measured at the same time point and under the same experimental conditions, one-way ANOVA was performed to determine the overall difference, followed by Tukey’s post hoc test if an overall difference was detected. This was accomplished with SPSS software. To compare the difference among regression lines, the regression coefficient, slopes, and intercepts were determined by the least-squares method by use of the Prism GraphPad program. The slope of the regression line is the efflux rate (%/min) presented with ses. Because both slopes and intercepts were compared, n-2 was used as the sample size for statistical analysis rather than n-1.

Acknowledgments

TM-BBB4 cells were provided by Dr. Tetsuya Terasaki at Tohoku University in Sendai, Japan. We thank Dr. Roger Laine (Louisiana State University) for helpful discussions, Ms. Loula Burton for editorial assistance, and other members of our BBB Group for help in the revision of the manuscript.

This work was supported by National Institutes of Health Grants NS45751 and NS46528 (to W.P.) and DK54880 (to A.J.K.).

Disclosure Statement: The authors have nothing to disclose.

Abbreviations

• BBB
• CNS
• CRHR
• db-cAMP
• αhCRH
• HEK
• IBMX
  3-isobutyl-1-methylxanthine.

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