Releasing Hormone Receptor

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Endocrinology 144(9):3860 –3871 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0028

Multiple Determinants for Rapid Agonist-Induced Internalization of a Nonmammalian GonadotropinReleasing Hormone Receptor: A Putative Palmitoylation Site and Threonine Doublet within the CarboxylTerminal Tail Are Critical ADAM J. PAWSON, STUART R. MAUDSLEY, JOHN LOPES, ARIEH A. KATZ, YUH-MAN SUN, JAMES S. DAVIDSON, AND ROBERT P. MILLAR Human Reproductive Sciences Unit, Medical Research Council (A.J.P., S.R.M., R.P.M.), Edinburgh, United Kingdom EH16 4SB; and Divisions of Chemical Pathology and Medical Biochemistry, University of Cape Town Medical School (A.J.P., J.L., A.A.K., Y.-M.S., J.S.D., R.P.M.), Cape Town 7925, South Africa The chicken GnRH receptor (cGnRH-R) differs from all mammalian GnRH-Rs in possessing a cytoplasmic carboxyl-terminal tail. We have previously demonstrated that the cGnRH-R undergoes more rapid agonist-induced internalization than the mammalian GnRH-Rs and requires the carboxyl-terminal tail for this process. To investigate the structural determinants mediating this rapid internalization, a series of mutant receptors was generated, including progressive truncations of the tail and substitution of serine and threonine residues with alanine. Truncation of the carboxyl-terminal tail to position 366 and then to position 356 resulted in a progressive attenuation of the rate and total extent of receptor internalization. However, truncation between positions 356 and 346 did not alter the kinetics of internalization further, whereas a further truncation to position 337 resulted in an additional marked reduction of internalization. We show that the

G

nRH IS A hypothalamic decapeptide that acts via its receptors on cells of the anterior pituitary to stimulate the release of both FSH and LH, and thus plays a pivotal role in reproduction. The receptor for GnRH is a member of the rhodopsin-like, G protein-coupled receptor (GPCR) family and couples to phospholipase C (PLC) activation via the Gq/11 family of G proteins (1). GPCRs are characterized structurally as seven transmembrane-spanning helixes linked by extracellular loops and intracellular loops and bearing an extracellular amino-terminal domain that may be glycosylated and a cytoplasmic carboxyl-terminal tail that may be palmitoylated. In general, the extracellular domains and/or transmembrane regions are involved in formation of the ligand binding pocket, whereas the cytoplasmic regions present sites for interactions with G proteins and other intracellular regulatory proteins (1, 2). The mammalian GnRH-Rs are unique among the rhodopAbbreviations: ␤-arr1, Wild-type ␤-arrestin-1; Bmax, maximal binding level; cGnRH-R, chicken GnRH receptor; CKII, casein kinase II; dyn-1, wild-type dynamin-1; FCS, fetal calf serum; GFP, green fluorescence protein; GPCR, G protein-coupled receptor; GRK, GPCR kinase; IPmax, maximal total inositol phosphate; m␤CD, methyl-␤-cyclodextrin; MDC, monodansylcadavarine; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C.

membrane-proximal Cys328 and the Thr369Thr370 doublet located in the distal carboxyl terminus play a critical role in mediating rapid internalization. We demonstrate that the cGnRH-R, when expressed in both COS-7 and HEK 293 cells, preferentially undergoes rapid agonist-induced internalization in a caveolae-like, dynamin-dependent manner. These conclusions are based on our observation that pretreatments with filipin and methyl-␤-cyclodextrin, agents that disrupt lipid rafts such as caveolae, and coexpression of dominantnegative dynamin-1 (K44A) and caveolin-1 (⌬1– 81) mutants, effectively inhibited rapid agonist-induced internalization. Furthermore, cGnRH-Rs appeared to be mobilized to the ␤arrestin- and clathrin-coated, vesicle-mediated endocytic pathway upon ␤-arrestin overexpression. (Endocrinology 144: 3860 –3871, 2003)

sin-like GPCR superfamily, because they completely lack a cytoplasmic carboxyl-terminal tail (2). In many GPCRs, this region has been demonstrated to play a crucial regulatory role in agonist-induced receptor phosphorylation, uncoupling, desensitization, internalization, and resensitization (3– 7). In keeping with these observations, the mammalian GnRH-R does not show rapid desensitization (8). In contrast to the mammalian GnRH-Rs, but in common with other GPCRs, the cloned nonmammalian GnRH-Rs all have a carboxyl-terminal tail (2, 9 –12). We have previously demonstrated that the chicken GnRH-R (10) exhibits rapid internalization kinetics and requires the carboxyl-terminal tail for this process (13). Furthermore, it has been reported that the catfish (14), Xenopus laevis (15, 16), and bullfrog (17) GnRH-Rs also have enhanced internalization kinetics compared with the mammalian GnRH-Rs. The importance of the carboxyl-terminal tail of the catfish GnRH-R for cell surface expression, ligand binding, and receptor phosphorylation and internalization has also been shown (18, 19). Moreover, addition of the carboxyl-terminal tail of the catfish GnRH-R to the rat GnRH-R results in an increased level of cell surface expression (20), and introduction of the carboxyl-terminal tail of the rat TRH receptor at the carboxyl terminus of the rat GnRH-R led to increased internalization (14). Other stud-

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Pawson et al. • Functional Significance of GnRH Receptor Carboxyl-Terminal Tail

ies have shown that the mammalian GnRH-Rs exhibit slow internalization kinetics (7, 14 –16, 21). These studies thus suggest that the carboxyl-terminal tail of nonmammalian GnRH-Rs plays a pivotal role in their function and subcellular trafficking. At least four pathways of agonist-induced internalization of GPCRs exist (22, 23), and they may be cell type specific. The classical GPCR internalization pathway involves GPCR kinases (GRKs), ␤-arrestin, clathrin-coated pits, and the GTPase dynamin and is exemplified by the ␤2-adrenergic receptor (3–7, 22, 23). The receptor is phosphorylated at specific serine and threonine residues by a GRK, followed by the binding of ␤-arrestin, whereupon the receptor is targeted to clathrin-coated pits for internalization. Dynamin is thought to be necessary for the scission of clathrin-coated vesicles from the plasma membrane (24). GPCRs have also been reported to internalize independently of both ␤-arrestin and dynamin or in pathways dependent on only one or the other, implying that GPCRs can undergo internalization via pathways distinct from clathrin-coated pits (3– 6, 22, 23). The localization of certain GPCRs to smooth noncoated membrane structures and vesicles (25, 26) suggests that GPCRs can use internalization pathways distinct from clathrincoated vesicles. Caveolae are flask-shaped, nonclathrincoated structures that have been implicated in the internalization of small molecules and certain GPCRs (27–33). In addition, dynamin has been implicated in caveolae function, although its exact functional role is not known. Dynamin may be responsible for the pinching off of caveolae from the plasma membrane (34, 35). We have previously reported that the chicken GnRH receptor (cGnRH-R) undergoes rapid agonist-induced internalization and requires the carboxyl-terminal tail for this process (13). In this study we investigate the role of the carboxyl-terminal tail of the cGnRH-R in relation to its rapid internalization and identify the key residues and mechanisms involved. It is known that COS-7 cells endogenously express low levels of ␤-arrestin, in contrast to the high levels present in HEK 293 cells (36). In this context we have sought to characterize the mechanisms involved in the rapid internalization of the cGnRH-R in COS-7 and HEK 293 cells by cotransfection of receptor constructs with wild-type and dominant-negative mutants of ␤-arrestin, dynamin, and caveolin and by the inhibition of clathrin-mediated and caveolar endocytosis.

Endocrinology, September 2003, 144(9):3860 –3871 3861

(Invitrogen), and the mutations were confirmed by automated sequencing (ABI PRISM, PE Applied Biosystems, Foster City, CA).

Cell culture and transient transfection Plasmid DNA for transient transfection was prepared using MaxiPrep columns (QIAGEN, Chatsworth, CA) according to the manufacturer’s instructions. COS-7 and HEK 293 cells were cultured as previously described for COS-1 cells (13) and transiently transfected using the SuperFect (QIAGEN) method according to the manufacturer’s instructions. Wild-type ␤-arrestin-1 (␤-arr1) and (319 – 418)␤-arr1 cDNAs were provided by Dr. J. L. Benovic, wild-type dynamin-1 (dyn-1) and dominant-negative (K44A)dyn-1 cDNAs were provided by Dr. M. G. Caron, and the green fluorescence protein (GFP)-tagged, dominantnegative caveolin-1 cDNA, which includes the deletion of residues 1– 81 [cav-1(⌬1– 81)], was provided by Dr. J. Eggermont. Pretreatments with monodansylcadavarine (MDC; Sigma-Aldrich Corp., St. Louis, MO), sucrose (Sigma-Aldrich Corp.), filipin (Sigma-Aldrich Corp.), and methyl-␤-cyclodextrin (m␤CD; Sigma-Aldrich Corp.) were performed 37 C for 30 min before receptor internalization assays.

Receptor binding assays Whole cell receptor binding assays used the 125I-[His5,d-Tyr6]GnRH analog (37). Transiently transfected COS-7 cells in 12-well culture plates were washed once with ice-cold HEPES/DMEM/10% fetal calf serum (FCS) and incubated for 5 h on ice in HEPES/DMEM/10% FCS with 105 cpm/well 125I-[His5,d-Tyr6]GnRH and varying concentrations of unlabeled [His5,d-Tyr6]GnRH. Cell monolayers were rapidly washed twice in ice-cold PBS and solubilized in 0.1 m NaOH, which was counted using a ␥-counter to determine the amount of bound radioligand. Nonspecific binding, consistently found to be less than 10% of total binding, was determined using vector-transfected (pcDNA1/amp) COS-7 cells and was subtracted from total binding to give specific binding.

Total inositol phosphate assays GnRH stimulation of total inositol phosphate production was previously described (38). Briefly, transiently transfected COS-7 cells were incubated with inositol-free DMEM containing 1% dialyzed heat-inactivated FCS and 0.5 ␮Ci/well myo-[3H]inositol (Amersham Pharmacia Biotech, Piscataway, NJ) for 48 h. Medium was removed, and the cells were washed with 1 ml buffer (140 mm NaCl, 20 mm HEPES, 4 mm KCl, 8 mm glucose, 1 mm MgCl2, 1 mm CaCl2, and 1 mg/ml BSA) containing 10 mm LiCl and incubated for 1 h at 37 C in 0.5 ml buffer containing 10 mm LiCl and GnRH agonist at the indicated concentration. Reactions were terminated by the removal of agonist and the addition of 1 ml ice-cold 10 mm formic acid, which was incubated for 30 min at 4 C. Total [3H]inositol phosphates were separated from the formic acid cell extracts on AG 1-X8 anion exchange resin (Bio-Rad Laboratories, Hercules, CA) and eluted with a 1 m ammonium formate/0.1 m formic acid solution. The associated radioactivity was determined by liquid scintillation counting.

Receptor internalization assays Materials and Methods Site-directed mutagenesis The cGnRH-R (10) was subcloned into pcDNA1/amp (Invitrogen, San Diego, CA). Mutations of the cGnRH-R carboxyl-terminal tail were introduced by a PCR-based method as previously described (13). For truncation mutants, Ser337, Ser346, Thr351, Asp356, and Ser366 were replaced by a stop codon, producing the truncated cGnRH-Rs (S337stop, S346stop, T351stop, D356stop, and S366stop). For point mutations, Ser337, Thr362Ser363, Ser366, Thr369Thr370, and Thr373 were mutated to alanine residues, producing single- or double-mutant cGnRH-R cDNAs (S337A, T362S363AA, S366A, T369T370AA, and T373A). A putative palmitoylation site (Cys328) was mutated to alanine, producing the C328A cGnRH-R cDNA. This mutation was additionally combined with the T369T370AA mutation to produce the C328AT369T370AA cGnRH-R cDNA. Mutant receptor cDNAs were subcloned into pcDNA1/amp

Receptor-mediated internalization of 125I-[His5,d-Tyr6 ]GnRH was determined by the acid-wash method as previously described for COS-1 cells (13). Briefly, transiently transfected COS-7 and HEK 293 cells in 12-well culture plates (poly-l-lysine coated for HEK 293 cells) were washed once with ice-cold HEPES/DMEM/10% FCS, then incubated on ice in HEPES/DMEM/10% FCS with 105 cpm/well 125I-[His5,d-Tyr6]GnRH for 5 h. Cells were rapidly warmed to 37 C for the indicated time periods, and internalization was stopped by placing the cells on ice and rapidly washing twice in 1 ml ice-cold PBS. Acid-sensitive, bound radioligand, representing cell surface-bound label, was removed by the addition of 1 ml ice-cold acid solution (50 mm acetic acid and 150 mm NaCl, pH 2.8) for 12 min. After removal of the acid wash, cells were solubilized with 1 ml 0.1 m NaOH to determine acid-resistant (internalized) radioligand content. Radioligand internalization was expressed as the percentage of total cell-associated label (acid-sensitive plus acidresistant) at each time point.

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Immunoprecipitation and Western blotting GFP-tagged cav-1(⌬1– 81) protein was immunoprecipitated (39) from cell lysates by overnight incubation with GFP-agarose slurry (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and washed. The immunoprecipitates were resolved by SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane (NEN Life Science Products, Boston, MA). Cav-1(⌬1– 81) protein was detected using antigoat GFP polyclonal antisera (Abcam, Cambridge, UK) and was visualized by enzyme-linked chemifluorescence (Amersham Pharmacia Biotech) and a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Results Characterization of GnRH binding and inositol phosphate production of wild-type and mutant cGnRH-Rs expressed in COS-7 cells

To investigate the functional role of the carboxyl-terminal tail in mediating rapid agonist-induced internalization of the cGnRH-R, the tail was truncated, or serines and threonines within the tail were mutated to alanines (Fig. 1). All of the mutant receptors were expressed at the cell surface and generated an inositol phosphate response when exposed to GnRH agonist (Table 1). Progressive truncation of the carboxyl-terminal tail resulted in decreased levels of receptor expression at the cell surface (as measured by whole cell binding of 125I-[His5,d-Tyr6]GnRH), with maximal binding levels (Bmax) ranging from 77 ⫾ 35% to 30 ⫾ 10% of wild-type cGnRH-R levels. The T373A, T369T370AA, S366A, T362S363AA, and S337A mutant receptors were all expressed at the cell surface, with Bmax levels ranging from 86 ⫾ 9% to 66 ⫾ 23% of wild-type levels. The affinities (estimated using the 50% inhibitory concentration) of the

FIG. 1. The carboxyl-terminal tail of the cGnRH-R. Schematic representation of the carboxyl-terminal tail of the cGnRH-R. Truncations are represented by lollypops at Ser337, Ser346, Thr351, Asp356, and Ser366. Those residues mutated to alanine are indicated.

mutant receptors were all within the nanomolar range and did not differ greatly from the wild-type receptor affinity (Table 1). With the exception of the S337stop (60 ⫾ 12%), S337A (73 ⫾ 8%), and T369T370AA (84 ⫾ 19%) cGnRH-R mutants, agonist stimulation of all remaining mutant receptors resulted in maximal total inositol phosphate (IPmax) levels that were higher than those seen for the wild-type receptor (Table 1). The lower 50% inhibitory concentration of the S337A mutant may be due to partial uncoupling of the receptor from Gq/11, which is suggested by the low IPmax. In contrast, although the IPmax was equally low in the S337stop mutant, its affinity was the same as that of the wild-type receptor. The higher 50% effective concentration of the S337stop mutant is most likely due to the lower expression of this receptor (Bmax ⬃30% of wild-type). The 50% effective concentrations of the mutant receptors were all (except for S337stop) in the nanomolar range and did not differ greatly from the wild-type value (Table 1). The relative levels of total inositol phosphate accumulation largely paralleled the maximal binding levels. The above data suggest that the length of the carboxyl-terminal tail of the cGnRH-R is important for efficient receptor expression at the cell surface, whereas specific serine and threonine residues located within the tail play a lesser role in expression. The decreased binding seen for the truncated mutants was due to decreased cell surface expression, rather than a perturbed ligand binding pocket caused by either truncation of the carboxyl-terminal tail or altered G protein coupling efficiencies, as the binding affinities (except for S337A) were unchanged. The decreased cell surface expression of the



TABLE 1. Summary of ligand binding and total inositol phosphate accumulation for wild-type and mutant cGnRH-Rs 125

Transfected construct

Wild type S366STOP D356STOP T351STOP S346STOP S337STOP T373A T369T370AA S366A T362S363AA S337A C328A C328AT369T379AA

I-[His5D-Tyr6]GnRH binding

Total inositol phosphate production

Bmax (% of wt)

IC50 (nM), [His5D-Tyr6]GnRH

IPmax (% of wt)

EC50 (nM), [Gln8]GnRH

100 77 ⫾ 35 45 ⫾ 27 55 ⫾ 28 61 ⫾ 28 30 ⫾ 10 81 ⫾ 13 86 ⫾ 9 75 ⫾ 15 85 ⫾ 6 66 ⫾ 23 69 ⫾ 12 250 ⫾ 43

2.8 ⫾ 1.4 4.7 ⫾ 1.0 5.2 ⫾ 2.5 5.3 ⫾ 2.4 3.7 ⫾ 1.7 2.4 ⫾ 1.6 3.7 ⫾ 1.2 4.3 ⫾ 3.7 5.4 ⫾ 3.9 4.6 ⫾ 1.4 0.34 ⫾ 0.1 5.2 ⫾ 2.7 4.6 ⫾ 0.6

100 147 ⫾ 33 191 ⫾ 53 158 ⫾ 27 136 ⫾ 27 60 ⫾ 12 132 ⫾ 16 84 ⫾ 19 143 ⫾ 26 134 ⫾ 36 73 ⫾ 8 98 ⫾ 7 132 ⫾ 14

5.5 ⫾ 4.1 3.8 ⫾ 3.7 9.1 ⫾ 5.6 7.4 ⫾ 5.2 2.9 ⫾ 1.8 16.6 ⫾ 6.5 2.8 ⫾ 0.2 1.4 ⫾ 0.1 1.2 ⫾ 0.1 1.0 ⫾ 0.1 1.7 ⫾ 1.1 2.6 ⫾ 1.7 5.9 ⫾ 1.5

Ligand binding parameters were determined from competition whole cell receptor binding assays in transfected COS-7 cells using 125I[His5,D-Tyr6]GnRH as tracer. Data represent the mean ⫾ SEM for two or three independent assays performed in duplicate. Total inositol phosphate production was determined in transfected COS-7 cells labeled with myo-[3H]inositol. Data represent the mean ⫾ SEM for two or three independent assays performed in duplicate.

truncated mutants may be due to either general structural instability of the receptor, leading to a global reduction of both nascent and plasma membrane-associated receptor, or a reduction in specific cell surface expression. Rapid agonist-induced internalization of cGnRH-R is mediated by a threonine-doublet at the carboxyl-terminal end of the cytoplasmic tail and a membrane-proximal cysteine residue

We have shown that the cGnRH-R undergoes rapid agonist-induced internalization and requires an intact carboxyl-terminal tail for this process (13). To more precisely localize the structural elements in the carboxyl terminus critical for the rapid internalization process, the cytoplasmic tail was progressively truncated (Fig. 1). The method used for measuring agonist-induced internalization discounts variations in receptor number due to decreased expression, because the data are presented as a percentage of the total agonist-bound receptors that are internalized at each time point measured. As the tail is progressively truncated, the mutant receptors undergo slower initial and decreased maximal levels of agonistinduced internalization (Fig. 2A). The most truncated S337stop mutant receptor exhibited a maximal steady state level of internalization only about 25% of that measured for the wild-type cGnRH-R after 90 min. This low level of internalization was similar to that of the wild-type human GnRH-R, which lacks a tail (13). There was little or no difference in both the initial and maximal levels of internalization of the S346stop, T351stop, and D356stop mutant receptors (internalization ⬃70% of wild-type), suggesting that the region between Ile345 and Asp356 does not contain critical elements for the rapid internalization of the cGnRH-R. We therefore targeted putative serine and threonine phosphorylation sites flanking this region for mutation to alanine residues (Fig. 1) and measured the abilities of these single and double mutant receptors to undergo agonist-induced internalization. With the exception of the T369T370AA receptor (internalization ⬃70% of wild-type), all of the remaining single and double mutants

showed little or no difference in both the initial and maximal levels of internalization (Fig. 2B). These results suggest that the Thr369Thr370 doublet plays a critical role in the rapid agonist-induced internalization of the cGnRH-R. This conclusion is further strengthened by the finding that that the D356stop, T351stop, and S346stop mutant receptors all internalized at approximately the same maximum steady state level as the T369T370AA mutant, indicating that the threonine residues at positions 369 and 370 are critical for rapid internalization. Interestingly, the S366stop mutant receptor, which does not contain Thr373, Thr369Thr370, or Ser366 residues, exhibited a maximum steady state level of internalization only about 10% lower than that measured for the wild-type receptor. This result is unexpected and difficult to interpret. It is possible that truncating the tail to Gly365 (i.e. the S366stop mutant), which now places Thr362 and Ser363 close to the carboxyl terminus of the receptor, enables them to fulfill the important role of the Thr369Thr370 doublet near the carboxyl terminus of the wild-type receptor. To investigate the functional role of the membrane-proximal cysteine residue in mediating rapid agonist-induced internalization of the cGnRH-R, Cys328 was mutated to alanine, alone and in combination with the T369T370AA mutation (i.e. the C328AT369T370AA mutation; Fig. 1). Both mutant receptors were expressed at the cell surface and generated an inositol phosphate response when exposed to GnRH agonist (Table 1). The ability of the C328A mutant receptor to internalize was reduced to similar initial and maximal levels as those seen for the T369T370AA mutant (Figs. 2B and 3). This result suggests that Cys328 is additionally important to the rapid agonist-induced internalization process of the cGnRH-R. The approximately 20% component of internalization that remains for the S337stop truncation mutant (Fig. 2A) may be mediated by residues in the region upstream of Ser337, of which Cys328 is one. This approximately 20% remaining component is similar to the 20 –25% inhibition of internalization observed for the C328A mutant receptor (Fig. 3). These results suggest that Cys328 is the most important residue in the tail region upstream of Ser337. Com-

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The cGnRH-R expressed in COS-7 cells undergoes rapid agonist-induced internalization via a ␤-arrestin- and clathrin-independent pathway

FIG. 2. Agonist-induced internalization of wild-type, truncated (A), and point-mutated (B) cGnRH-Rs expressed in COS-7 cells. Receptor constructs (1.5 ␮g cDNA/well of a 12-well plate) A) wildtype (f), S366stop (Œ), D356stop (), T351stop (F), S346stop (⽧), and S337stop (䡺); and B) wild-type (f), T373A (Œ), T369T370AA (), T369T370EE (F), S366A (⽧), T362S363AA (䡺), and S337A (‚) were expressed in COS-7 cells. Agonist-induced internalization was performed as described in Materials and Methods. The results shown are the mean ⫾ SEM of six independent assays performed in duplicate.

bination of the Cys328A and Thr369Thr370AA mutations led to a mutant receptor that internalized slightly lower than both the C328A or T369T370AA mutant receptors (Figs. 2A and 3). The fact that the creation of the double mutant did not produce an additive effect suggests distinct roles for Cys328 and the Thr369Thr370 doublet in the rapid agonist-induced internalization process. Interestingly, the C328AT369T370AA mutant receptor showed a 2.5-fold higher expression level at the cell surface compared with the wild-type receptor (Table 1). The higher IPmax value (IPmax ⬃132% of wild type) of the C328AT369T370AA mutant is most likely due to the higher expression of this receptor (Bmax ⬃250% of wild type).

We further sought to characterize the preferred pathway of internalization of the wild-type cGnRH-R in COS-7 cells. Thus, we examined the effect of wild-type ␤-arrestin-1 (␤arr1) and dynamin coexpression, and/or pretreatments or coexpression with disrupters of clathrin-coated pit function and caveolae formation on the internalization process. In subsequent receptor internalization assays, the internalization levels after 5 min are shown (Figs. 4– 6, and 7A). To examine the effect of ␤-arrestin on cGnRH-R internalization (Fig. 4), the cGnRH-R construct was cotransfected with vector (pcDNA1/amp) as a control, wild-type ␤-arr1, dominantnegative (319 – 418)␤-arr1, or both ␤-arr1 and (319 – 418)␤arr1. Coexpression with ␤-arr1 led to increased cGnRH-R internalization (Fig. 4, u). This increase in internalization under conditions of ␤-arr1 overexpression will henceforth be referred to as ␤-arr1-promoted internalization. Coexpression of the dominant-negative (319 – 418)␤-arr1 construct was effective in blocking the enhancing effect of ␤-arr1 overexpression on internalization (Fig. 4, p), indicating that the encoded protein was expressed and functional in the negative sense. Despite this, (319 – 418)␤-arr1 coexpression alone had virtually no effect on internalization (Fig. 4, 䡺), indicating that cGnRH-Rs are internalized via a ␤-arr1independent pathway in COS-7 cells unless ␤-arr1 is overexpressed. This lack of effect is consistent with the reported low levels of endogenously expressed ␤-arr1 in COS-7 cells (36). Pretreatment with disrupters of clathrin-coated pit function, MDC (40) and hypertonic sucrose (41), failed to inhibit internalization (Fig. 5) even at later time points (data not shown), whereas MDC and sucrose pretreatments were effective at blocking ␤-arr1-promoted internalization (Fig. 5). This is consistent with a role for clathrin-coated pit formation in the ␤-arr1-dependent internalization pathway. MDC pretreatments showed similar effects when tested at concentrations from 50 – 400 ␮m for periods varying from 30 –90 min (data not shown). The above results suggest that in COS-7 cells the wild-type cGnRH-R preferentially undergoes rapid agonist-induced internalization through a pathway that is independent of both ␤-arr1 and clathrin-coated pits. However, under conditions of ␤-arr1 overexpression, the cGnRH-R can be mobilized or recruited to internalize via a ␤-arr1- and clathrin-mediated pathway. The cGnRH-R expressed in COS-7 cells undergoes rapid agonist-induced internalization via a dynamin- and caveolae-mediated pathway

To examine the effect of dynamin on cGnRH-R internalization (Fig. 6), the cGnRH-R construct was cotransfected with vector (pcDNA1/amp) as a control, wild-type dynamin-1 (dyn-1), dominant-negative (K44A)dyn-1, or both wild-type ␤-arr1 and (K44A)dyn-1. Coexpression with wild-type dyn-1 had no effect on internalization (data not shown), whereas coexpression with the dominant-negative (K44A)dyn-1 construct inhibited internalization (Fig. 6, 䡺), suggesting the utilization of a dynamin-dependent pathway. Furthermore,



FIG. 3. Agonist-induced internalization of wild-type, C328A, and C328AT369T370AA cGnRH-Rs expressed in COS-7 cells. Receptor constructs (1.5 ␮g cDNA/well of a 12-well plate) wild-type (f), C328A (Œ), and C328AT369T370AA () were expressed in COS-7 cells. Agonist-induced internalization was performed as described in Materials and Methods. The results shown are the mean ⫾ SEM of three independent assays performed in triplicate.

FIG. 4. Agonist-induced internalization of wild-type cGnRH-Rs expressed in COS-7 cells or coexpressed with wild-type and/or a dominant-negative mutant of ␤-arr1. A wild-type cGnRH-R cDNA construct (0.75 ␮g cDNA/well of a 12-well plate) was coexpressed with 0.75 ␮g of vector (pcDNA1/amp; control; f), dominant-negative (319 – 418)␤-arr1 (䡺), wild-type ␤-arr1 (u), or both wild-type ␤-arr1 and dominant-negative (319 – 418)␤-arr1 (p). Agonist-induced internalization was performed as described in Materials and Methods. The results shown are the mean ⫾ SEM of three or four independent assays performed in triplicate.

the coexpression of receptors with both wild-type ␤-arr1 and dominant-negative (K44A)dyn-1, revealed that ␤-arr1promoted internalization was blocked in the presence of coexpressed (K44A)dyn-1 (Fig. 6, p). Pretreatment with filipin and m␤CD, disrupters of caveolae formation (42), led to an inhibition of cGnRH-R internal-

FIG. 5. The effect of pretreatment with disrupters of clathrin-coated pits on agonist-induced internalization of wild-type cGnRH-Rs expressed in COS-7 cells in the presence or absence of ␤-arr1 overexpression. The wild-type cGnRH-R cDNA construct (0.75 ␮g cDNA/well of a 12-well plate) was coexpressed with 0.75 ␮g of vector (pcDNA1/ amp; control; f), wild-type ␤-arr1 (䡺), vector and MDC (400 ␮M) pretreatment (u), wild-type ␤-arr1 and MDC (400 ␮M) pretreatment (z), vector and sucrose (0.45 M) pretreatment (p), or wild-type ␤-arr1 and MDC (400 ␮M) pretreatment (`). Agonist-induced internalization was performed as described in Materials and Methods. Pretreatments with MDC and sucrose were performed for 30 min at 37 C before the receptor internalization assay. The results shown are the mean ⫾ SEM of three or four independent assays performed in triplicate.

ization (Fig. 7A, 䡺, and Fig. 7B). In addition, filipin pretreatment was apparently able to effectively inhibit ␤-arr1promoted internalization (Fig. 7A, p). Filipin pretreatments were performed at concentrations from 0.5–5 ␮g/ml for time periods varying from 30 –90 min with no alteration in the level of inhibition of internalization being evident (data not

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our finding that pretreatment of wild-type cGnRH-R expressing HEK 293 cells with MDC or hypertonic sucrose had virtually no effect on internalization (Fig. 8B). However, coexpression with dominant-negative (319 – 418)␤-arr1 clearly retarded the initial rate of agonist-induced internalization, but had no effect on the maximal steady state level that wild-type cGnRH-Rs can reach in HEK 293 cells (Fig. 8B). In contrast, both coexpression with dominant-negative (K44A)dyn-1 and pretreatment with filipin effectively inhibited internalization of wild-type cGnRH-Rs expressed in HEK 293 cells, reducing both the initial rate and the maximal steady state level (Fig. 8C). These results suggest that despite the high background of endogenous ␤-arr1 expression, the wild-type cGnRH-R preferentially undergoes rapid agonistinduced internalization in caveolae in a dynamin-dependent manner in HEK 293 cells. Discussion

FIG. 6. Agonist-induced internalization of wild-type cGnRH-Rs expressed in COS-7 cells or coexpressed with dominant-negative (K44A)dyn-1 in the presence or absence of ␤-arr1 overexpression. The wild-type cGnRH-R cDNA construct (0.75 ␮g cDNA/well of a 12-well plate) was coexpressed with 0.75 ␮g of vector (pcDNA1/amp; control; f), dominant-negative (K44A)dyn-1 (䡺), wild-type ␤-arr1 (u), or wildtype ␤-arr1 and dominant-negative (K44A)dyn-1 (p). Agonist-induced internalization was performed as described in Materials and Methods. The results shown are the mean ⫾ SEM of three or four independent assays performed in triplicate.

shown). The m␤CD pretreatments were performed at 10 ␮m for 30 min. Caveolin-1 is the principal structural protein of caveolae membranes (43). Coexpression of a dominantnegative mutant [cav-1(⌬1– 81)] of caveolin-1, previously shown to disrupt the formation of caveolae lipid rafts (44), led to a significant inhibition of cGnRH-R internalization (Fig. 7D). We transiently transfected the GFP-tagged cav1(⌬1– 81) construct into COS-7 cells and assessed its expression relative to mock-transfected cells by Western blot analysis with specific anti-GFP antisera (Fig. 7C). These results suggest that in COS-7 cells, the wild-type cGnRH-R preferentially undergoes rapid agonist-induced internalization in caveolae in a dynamin-dependent manner. The cGnRH-R expressed in HEK 293 cells undergoes rapid agonist-induced internalization via a dynamin- and caveolae-mediated pathway

We were interested to compare the pathways of rapid agonist-induced wild-type cGnRH-Rs when expressed in COS-7 and HEK 293 cells, based on the significant differences in the background endogenous expression of ␤-arr1 in these two cell lines (36). A 90-min course of internalization of transiently expressed wild-type receptor revealed that although the initial rates of internalization in the two cell types appeared to be very similar, the maximal steady state level was about 30% lower in HEK 293 cells than in COS-7 cells (Fig. 8A). This unexpected result was further complicated by

This study has investigated the functional role of the carboxyl-terminal tail of the cGnRH-R in relation to its rapid agonist-induced internalization and identified the preferred pathway of internalization of the cGnRH-R in COS-7 cells. Progressive truncation of the carboxyl-terminal tail identified two critical regions, upstream of Ser346 and downstream of Asp356. Phosphate acceptors within the carboxyl-terminal tails of many GPCRs have previously been shown to play an important role in their internalization (3–7, 22, 23). Thus, the serine and threonine residues in these two regions were mutated to alanine to investigate the role of these putatively phosphorylated residues in the agonist-induced internalization process. We demonstrated that a threonine doublet (Thr369Thr370) located near the carboxyl terminus and the membrane-proximal cysteine residue (Cys328) are the two major contributors in the rapid internalization process of the cGnRH-R, whereas other carboxyl-terminal tail residues may play a lesser role. Our results showed that the cGnRH-R expressed in COS-7 cells undergoes rapid agonist-induced internalization in a ␤-arr1- and clathrin-coated pit-independent manner, based on the finding that neither a dominantnegative mutant of ␤-arr1 nor disrupters of clathrin-coated pit function [MDC (40), and hypertonic sucrose (41)] were effective at inhibiting internalization, except under conditions of ␤-arr1 overexpression. These findings were not surprising in light of the fact that COS-7 cells have been shown to endogenously express low levels of ␤-arrestin (36). Despite this, the cGnRH-R undergoes rapid agonist-induced internalization in COS-7 cells. To further characterize the internalization pathway of the cGnRH-R expressed in COS-7 cells, we employed the dominant-negative (K44A)dyn-1 mutant and disrupters of caveolar structure and function [filipin (42), m␤CD (45), and a dominant-negative caveolin-1 mutant (44)]. Our data demonstrated that the cGnRH-R preferentially undergoes rapid agonist-induced internalization in a dynamin- and caveolaedependent manner, based on the finding that internalization is inhibited in the presence of dominant-negative (K44A)dyn-1 and cav-1(⌬1– 81) overexpression, and pretreatments with filipin and m␤CD. We observed that under conditions of ␤-arr1 overexpres-



FIG. 7. The effect of disrupters of caveolae formation on agonist-induced internalization of wild-type cGnRH-Rs expressed in COS-7 cells. A, The wild-type cGnRH-R cDNA construct (0.75 ␮g cDNA/well of a 12-well plate) was coexpressed with 0.75 ␮g of vector (pcDNA1/amp; control; f), vector and filipin (5 ␮g/ml) pretreatment (䡺), wild-type ␤-arr1 (u), or wild-type ␤-arr1 and filipin (5 ␮g/ml) pretreatment (p). The results shown are the mean ⫾ SEM of three or four independent assays performed in triplicate. B, The wild-type cGnRH-R cDNA construct (1.5 ␮g cDNA/well of a 12-well plate) was expressed in COS-7 cells untreated (f), filipin (5 ␮g/ml) pretreatment (Œ), m␤CD (10 ␮M) pretreatment (). The results shown are the mean ⫾ SEM of three independent assays performed in triplicate. C, Depicts an anti-GFP immunoblot of anti-GFP immunoprecipitates from COS-7 cells expressing either vector or GFP-tagged dominant-negative caveolin-1 (⌬1– 81) cDNA, as described in Materials and Methods. D, The wild-type cGnRH-R cDNA construct (0.2 ␮g/well of a 12-well plate) was coexpressed in COS-7 cells with 1.8 ␮g of vector (pcDNA1/amp; control; f), or dominant-negative caveolin-1 (⌬1– 81) cDNA (‚). The results shown are the mean ⫾ SEM of three independent assays performed in triplicate. Agonist-induced internalization was performed as described in Materials and Methods. Pretreatments with filipin and m␤CD were performed for 30 min at 37 C before the receptor internalization assay.

sion, where cGnRH-Rs were additionally mobilized or recruited to the clathrin-coated pit-mediated internalization machinery, that filipin pretreatment was apparently effective at inhibiting ␤-arr1-promoted internalization (Fig. 7A, p). It could be argued from the above result that the effect of filipin is nonspecific. However, this result more likely depicts the filipin-mediated inhibition of the caveolar component of internalization, and that the remaining fraction is, in fact, the filipin-resistant internalization of those receptors that have been recruited to the ␤-arrestin-dependent pathway by

␤-arr1 overexpression (i.e. the ␤-arr1-promoted internalization component). Furthermore, coexpression with both wildtype ␤-arr1 and dominant-negative (K44A)dyn-1 (Fig. 6, p) revealed that in addition to inhibition of the ␤-arr1-promoted internalization component, (K44A)dyn-1 was effective at inhibiting the caveolar component as well. This result clearly implicates dynamin function in both clathrin- and caveolarmediated internalization pathways. Studies of the internalization pathways used by mammalian and nonmammalian GnRH-Rs show that the rat

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FIG. 8. Agonist-induced internalization of wild-type cGnRH-Rs expressed in HEK 293 cells in the absence or presence of coexpressed dominantnegative (319 – 418)␤-arr1 or (K44A)dyn-1 or pretreatment with disrupters of either clathrin-coated pits or caveolae. A, Comparison of agonist-induced internalization of wild-type cGnRH-Rs expressed in either COS-7 (䡺) or HEK 293 cells (f). B, The wild-type cGnRH-R cDNA construct (0.75 ␮g/well of a 12-well plate) was coexpressed in HEK 293 cells with 0.75 ␮g of vector (pcDNA1/amp; control; f), dominant-negative (319 – 418)␤-arr1 (Œ), vector (pcDNA1/amp) and MDC (400 ␮M) pretreatment (), or vector (pcDNA1/amp) and sucrose (0.45 M) pretreatment (⽧). C, The wild-type cGnRH-R cDNA construct (0.75 ␮g/well of a 12-well plate) was coexpressed in HEK 293 cells with 0.75 ␮g of vector (pcDNA1/amp; control; f), dominant-negative (K44A)dyn-1 (‚), or vector (pcDNA1/amp) and filipin (5 ␮g/ml) pretreatment (ƒ). Agonist-induced internalization was performed as described in Materials and Methods. The results shown are the mean ⫾ SEM of three independent assays performed in triplicate.

GnRH-R, which lacks a cytoplasmic tail, internalizes in a ␤-arrestin-independent manner, but probably via a clathrindependent mechanism (21), and in a ␤-arrestin-independent pathway that is dynamin dependent (46). Although these findings may appear to be inconsistent, it is cogent that a lack of a carboxyl-terminal domain in the mammalian GnRH-Rs probably confers their ␤-arrestin independence, at least as far as internalization is concerned. Furthermore, it has been shown that the human GnRH-R undergoes internalization via a clathrin-mediated pathway, but in a dynamin-independent manner (16). A study characterizing agonistinduced internalization of the catfish GnRH-R (19) identified a serine residue in the carboxyl-terminal tail that is phosphorylated and may function as a ␤-arrestin binding site. The conclusions of this study were based on the finding that

substitution of this serine residue with alanine (or deleting the last 12 amino acids of the tail, which includes this serine) resulted in mutant receptors whose impaired internalization was not rescued by ␤-arrestin overexpression in COS-7 cells (19). Furthermore, coexpression of the dominant-negative (319 – 418)␤-arrestin with the catfish GnRH-R in COS-7 cells produced a slight inhibition of the steady state level internalization (46). A recent study has characterized the internalization pathways of the three bullfrog GnRH-R subtypes (17). The bullfrog type II GnRH-R showed the most rapid rate and highest extent of internalization among the three receptors. Furthermore, internalization of the bullfrog type I GnRH-R was shown to be both ␤-arrestin and dynamindependent, whereas bullfrog type II and III GnRH-Rs internalize via a pathway that is ␤-arrestin independent, but dy-


namin dependent (17), similar to the pathway used by the chicken GnRH-R (present study). It is interesting to note that the last eight residues of the carboxyl-terminal tails of the chicken and bullfrog type II GnRH-Rs are surprisingly similar (GTTVNTVC for chicken, ATTVQSVF for bullfrog type II). The significance of this similarity is unclear, but may point to the importance of the threonine doublet identified here as critical for cGnRH-R internalization. The apparent propensity of the cGnRH-R for undergoing rapid agonist-induced internalization via caveolae in a dynamin-dependent manner and independently of ␤-arr1 and clathrin-coated vesicles is not restricted to COS-7 cells. Interestingly, the same internalization pathway is preferred in HEK 293 cells, which endogenously express about 70% more ␤-arrestin than COS-7 cells (36). However, the maximal steady state level of internalization of the wild-type cGnRH-R was roughly 30% lower when expressed in HEK 293 cells than in COS-7 cells. It is interesting to note that the maximum steady state level of internalization of the wildtype catfish GnRH-R is much lower in COS-7 cells than in HEK 293 cells (19), underpinning the importance of ␤-arrestin in the internalization pathway of the catfish GnRH-R. Clearly, both properties of a receptor and the cell type in which a receptor is expressed will determine the preferred pathway of agonist-induced internalization. It is therefore interesting to speculate about what, if any, the properties of the cGnRH-R are which make this receptor internalize preferentially in caveolae. We have shown that the presence of the carboxyl-terminal tail, the membrane-proximal Cys328, the Thr369Thr370 doublet are essential for rapid internalization of this receptor. Is it possible that these are the determinants for preferred internalization via caveolae? A number of studies have reported caveolae-mediated internalization of GPCRs, including the ETA endothelin receptor (28), the cholecystokinin receptor (33), the B2 bradykinin receptor (29, 32, 47), and the m2 muscarinic receptor (30). The m2 muscarinic receptor was previously shown to preferentially internalize in a ␤-arrestin-independent internalization pathway, but could be recruited to the ␤-arrestindependent pathway by overexpression of ␤-arrestin (48). Furthermore, the m1, m3, and m4 muscarinic receptors internalize via a ␤-arrestin-independent, but dynamin-dependent, internalization pathway (49, 50), the same pathway used by the rat GnRH-R (21, 46). Internalization of the B2 bradykinin receptor has been shown to proceed independently of both ␤-arrestin and dynamin, providing further support for its utilization of a caveolae-mediated pathway (47). Most of the GPCRs to date shown to internalize via caveolae are coupled to Gq/11 and PLC activation. Apart from this common feature, there are no other apparent similarities between these GPCRs that could explain their preference for internalization via caveolae. To date, no receptor-specific elements, structural or sequence motifs, have been demonstrated to particularly target a GPCR to caveolae or promote its internalization via caveolae. It has been hypothesized that fatty acylation (palmitoylation and/or myristoylation) could function to target certain signaling molecules and cell surface receptors to caveolae compartments (43). Furthermore, many caveolar components share a common motif for dual acyla-


tion by palmitate and myristate at their amino termini (51). In this context, we note that the carboxyl-terminal tail of the cGnRH-R may be palmitoylated at the membrane-proximal Cys328 residue. Most GPCRs posses a putative palmitoylation site in the carboxyl-terminal tail proximal to the seventh transmembrane-spanning helix. Amino acid sequence comparison of this region of the cGnRH-R with that of the ␤2adrenergic receptor [known to be palmitoylated at Cys341 (52)] reveals a very similar sequence pattern, suggesting that should palmitoylation of Cys328 occur, this modification could serve to target the cGnRH-R to caveolae for rapid agonist-induced internalization. In addition to the lack of identification of any sequence or structural motif being demonstrated to specifically target GPCRs to caveolae, the differences in dynamin dependency for caveolae-mediated internalization is a further complication (Refs. 31, 34, 35, and 47 and this study). It appears that the involvement of ␤-arrestin is not a prerequisite for internalization via a clathrin-mediated and dynamin-dependent pathway, and that the involvement of dynamin is not a prerequisite for internalization via a caveolae-mediated pathway. Alternatively, the pathways mediated by clathrincoated pits and caveolae may be interlinked or positioned in tandem as suggested in a recent study (47). Certain receptors may therefore associate with clathrin-coated pits subsequent to their redistribution in caveolae. This scenario could explain the findings of a study demonstrating that rat GnRH-Rs colocalized with transferrin receptors (known to internalize via the clathrin-coated vesicle pathway), even though rat GnRH-R internalization is apparently ␤-arrestin-independent (21). Recently, a study demonstrated that ␤2-adrenergic receptors, which are known to internalize via a ␤-arrestindependent, clathrin-mediated pathway, are initially localized within caveolae in neonatal myocytes (53). This localization was shown to be essential for signaling, which was disrupted by filipin treatment (53). As activation of ␤2adrenergic receptors leads to their rapid depalmitoylation and hyperphosphorylation (54), it is conceivable that this event could be the trigger to allow receptors to bind ␤arrestin and to be targeted from caveolae to the clathrinmediated internalization pathway. Recently, palmitoylationdeficient mutant LH/CG receptors were demonstrated to undergo enhanced internalization and appeared to be more prone to ␤-arrestin-dependent, clathrin-mediated internalization compared with wild-type receptors (55). In contrast, our C328A cGnRH-R mutant undergoes a markedly reduced level of internalization compared with the wild type. The above scenarios lend support to the idea that caveolae may be required by certain GPCRs to promote the initial formation of ligand receptor-G protein complexes and subsequent second messenger production by concentrating and increasing the proximity of the various components involved. Indeed, many species of G proteins, including Gq/11, have been shown to be enriched in caveolae (43, 56). There is evidence to suggest that the initial signaling events of other GPCRs coupled to Gq/11 and PLC activation may be predominantly restricted to caveolae (29). After this initial association in caveolae, certain receptors (e.g. ␤2-adrenergic receptor) may then be targeted to clathrin-coated pits for internalization, whereas others (e.g. cGnRH-R, presented here) may remain

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in caveolae and undergo internalization via these compartments. Dynamin has certainly been implicated in caveolaemediated endocytosis and may function in the scission of caveolae to form vesicles (34, 35). Furthermore, it has been shown that dynamin mediates caveolar internalization of muscarinic acetylcholine receptors (31). In the current model for the regulation of archetypal agonist-activated GPCRs, the receptor must first be phosphorylated at specific serine and/or threonine residues, which promotes ␤-arrestin binding. This results in rapid desensitization of the receptor by uncoupling it from its cognate G protein. The receptor is then targeted to clathrincoated pits for internalization (3–7, 22, 23). A number of kinases have been implicated in GPCR regulation, including GRKs, protein kinase C (PKC), protein kinase A (PKA), and casein kinase II (CKII). The consensus sequence motifs for GRKs have not yet been clearly defined (although serine and/or threonine residues within an acidic environment are required), whereas those for PKC [(S/T)X(R/K)], PKA [(R/ K)X(S/T)], and CKII [(S/T)XX(D/E)] have. The threonine doublet identified in this study does not lie in the consensus sequence motifs for PKC-, PKA-, or CKII-mediated phosphorylation, although the carboxyl-terminal tail of the cGnRH-R does contain putative PKC and CKII phosphorylation sites. These sites are, however, located in the region identified here as containing no elements essential for the rapid internalization of this receptor. This implies that PKCand CKII-mediated phosphorylation of the cGnRH-R plays no apparent role in its rapid internalization, but may well be essential for other aspects of cGnRH-R regulation, such as desensitization and uncoupling, and resensitization. A recent study has suggested that CKII sites present in the carboxyl-terminal tail of the TRH receptor or introduced by mutation into the tail of the catfish GnRH-R play a role in ␤-arrestin dependency for regulation of these receptors (57). In conclusion, our data clearly demonstrate that there can be fundamental differences with respect to receptor trafficking function when deletion mutagenesis is compared with site-directed mutagenesis. This demonstrates to some extent a useful precedent, in that considering a truncated and therefore grossly distinct receptor, comparable to a point-mutated receptor, may be an oversimplification of the situation. We have identified a threonine doublet (Thr369Thr370) and a cysteine residue (Cys328) that are critical for rapid agonistinduced internalization of the cGnRH-R. Although the mechanism by which the threonine doublet promotes rapid internalization is unclear, it is possible that it serves a structural role rather than a site for protein-protein interaction. Palmitoylation of Cys328 may serve to target the cGnRH-R to caveolae microdomains for signaling and internalization. Whether expressed in COS-7 or HEK 293 cells, the cGnRH-R preferentially internalizes via caveolae in a dynamin-dependent manner, but may be recruited to the ␤-arrestin- and clathrin-coated pit-dependent internalization pathway by overexpression of ␤-arrestin in COS-7 cells. Finally, we provide evidence to support previous studies suggesting that clathrin-coated pits and caveolae are interlinked or positioned in tandem rather than existing as two mutually exclusive cell surface microdomains.

Acknowledgments We are grateful to Robin Sellar and Nicola Miller for their excellent technical assistance. Received January 7, 2003. Accepted June 9, 2003. Address all correspondence and requests for reprints to: Prof. Robert P. Millar, Human Reproductive Sciences Unit, Medical Research Council, University of Edinburgh Chancellor’s Building, 49 Little France Crescent, Edinburgh, United Kingdom EH16 4SB. E-mail: [email protected]. This work was supported by the Wellcome Trust (to A.J.P., Grant 060257; and A.A.K.), the Medical Research Council of South Africa, the Medical Research Council of the United Kingdom, the Roche Research Foundation, the Loewenstein Trust, the Foundation for Research Development, the Harry Crossley Foundation, the Fogarty Foundation of NIH, and the University of Cape Town.

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