Regulation of melanocortin-4 receptor signaling: agonist-mediated desensitization and internalization

Disruption of the hypothalamic melanocortin-4 receptor (MC4R) pathway results in obesity both in humans and rodents, demonstrating a crucial role for hypothalamic MC4Rs in the regulation of energy homeostasis. Because even haploinsufficiency of the MC4R gene can cause obesity in humans and mice, subtle changes in receptor numbers or signaling are likely to impact upon the regulation of food intake and energy expenditure. Little is known about the intracellular regulation of MC4R signaling. Using GT1-7 cells, we show for the first time that the MC4R undergoes ligand-mediated desensitization. We then addressed the possible mechanisms underlying the desensitization using HEK293 and COS-1 cells transfected with hemagglutinin-tagged human MC4R. Preexposure of GT1-7 cells that express endogenous MC4R to the agonist for MC4R, alpha-melanocyte-stimulating hormone, resulted in impaired cAMP formation to a second challenge of alpha-melanocyte-stimulating hormone. The desensitization of MC4R was accompanied by time-dependent internalization of the receptor in HEK293 cells, which was partly inhibited by pretreatment with a specific protein kinase A (PKA) inhibitor, H89. In COS-1 cells, overexpression of dominant-negative G protein-coupled receptor kinase (GRK) 2-K220R partly inhibited the agonist-mediated internalization of MC4R, whereas it did not in HEK293 cells. Overexpression of dominant-negative mutants of beta-arrestin1-V53D and dynamin I-K44A prevented agonist-mediated internalization of MC4R. Mutagenesis studies revealed that Thr312 and Ser329/330 in the C-terminal tail are potential sites for PKA and GRK phosphorylation and may play an essential role in the recruitment of beta-arrestin to the activated receptor. Our data demonstrate that, through PKA-, GRK-, beta-arrestin-, and dynamin-dependent processes, MC4R undergoes internalization in response to agonist, thereby providing novel insights into the regulation of MC4R signaling.     

Agonist activated adrenocorticotropin receptor internalizes via a clathrin-mediated G protein receptor kinase dependent mechanism

The physiological effects of the pituitary hormone, adrenocorticotropic hormone (ACTH) on the adrenal are mediated by the melanocortin 2 receptor (MC2R), a G protein coupled receptor (GPCR) that signals via adenylate cyclase to elevate intracellular cyclic AMP (cAMP) levels. The function and expression of the receptor is likely to be a major determinant of the response to ACTH. Following repeated stimulation, the cAMP signal is diminished or desensitized. Prolonged desensitization may involve internalization of the receptor. Internalization may occur by at least two mechanisms--receptor mediated endocytosis via clathrin-coated pits and by caveolae mediated internalization. The mode of internalization for the endogenous MC2R in Y1 cells was determined using radiolabelled ACTH. Treatment of Y1 cells with hypertonic sucrose or with concanavalin A, which inhibit clathrin-mediated endocytosis, blocked internalization. Filipin and nystatin, which inhibit caveolae formation, did not influence internalization. A dominant negative GRK2 inhibited internalization whilst the protein kinase A (PKA) consensus site mutant MC2R (S208A) internalized normally. However, dominant negative V53D beta-arrestin-1 did not inhibit ACTH internalization in Y1 cells. In conclusion, it appears that the MC2R in Y1 cells internalizes by a G protein coupled receptor kinase (GRK) dependent clathrin-coated pit mechanism.     

Beta-Arrestin-1 mediates glucagon-like peptide-1 signaling to insulin secretion in cultured pancreatic beta cells

Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted from enteroendocrine L cells and potentiates glucose-dependent insulin secretion in pancreatic beta cells. Recently the GLP-1 receptor (GLP-1 R) has been a focus for new anti-diabetic therapy with the introduction of GLP-1 analogues and DPP-IV inhibitors, and this has stimulated additional interest in the mechanisms of GLP-1 signaling. Here we identify a mechanism for GLP-1 action, showing that the scaffold protein beta-arrestin-1 mediates the effects of GLP-1 to stimulate cAMP production and insulin secretion in beta cells. Using a coimmunoprecipitation technique, we also found a physical association between the GLP-1 R and beta-arrestin-1 in cultured INS-1 pancreatic beta cells. beta-Arrestin-1 knockdown broadly attenuated GLP-1 signaling, causing decreased ERK and CREB activation and IRS-2 expression as well as reduced cAMP levels and impaired insulin secretion. However, beta-arrestin-1 knockdown did not affect GLP-1 R surface expression and ligand-induced GLP-1 R internalization/desensitization. Taken together, these studies indicate that beta-arrestin-1 plays a role in GLP-1 signaling leading to insulin secretion, defining a previously undescribed mechanism for GLP-1 action.     

G protein-coupled receptor kinase 2 involvement in desensitization of corticotropin-releasing factor (CRF) receptor type 1 by CRF in murine corticotrophs

Hypothalamic CRF stimulates synthesis and secretion of ACTH via CRF receptor type 1 (CRFR1) in the anterior pituitary gland. After agonist-activated stimulation of receptor signaling, CRFR1 is down-regulated and desensitized. Generally, it is thought that G protein-coupled receptors may be desensitized by G protein-coupled receptor kinases (GRKs). However, the role of GRKs in corticotropic cells has not been determined. In this study we focused on involvement of GRKs in desensitization of CRFR1 by CRF in corticotropic cells. We found that GRK2 (but not GRK3) mRNA and protein were expressed in rat anterior pituitary cells and AtT-20 cells (a line of mouse corticotroph tumor cells). To determine the role of GRK2 in CRF-induced desensitization of CRFR1 in mouse corticotrophs, AtT-20 cells were transfected with a dominant-negative mutant GRK2 construct. CRF desensitized the cAMP-dependent response by CRFR1. Desensitization of CRFR1 by CRF was significantly less in AtT-20 cells transfected with the dominant-negative mutant GRK2 construct compared with desensitization in control (an empty vector-transfected) AtT-20 cells. Furthermore, pretreatment with a protein kinase A inhibitor also partially blocked desensitization of CRFR1 by CRF. These results suggest that GRK2 is involved in CRF-induced desensitization of CRFR1 in AtT-20 cells, and the protein kinase A pathway may also have an important role in desensitization of CRFR1 by CRF seen in corticotropic cells.     

Effect of hypothyroidism on G protein-coupled receptor kinase 2 expression levels in rat liver, lung, and heart

GRK2 is a member of the G protein-coupled receptor kinase family that phosphorylates the activated form of beta-adrenergic and other G protein-coupled receptors and plays an important role in their desensitization and modulation. Alterations in thyroid hormone levels have been reported to lead to important changes in adrenergic receptor responsiveness and signaling in a variety of tissues. In this context, we have explored the effects of experimental hypothyroidism on GRK2 protein levels in rat heart, lung, and liver using a specific antibody. Hypothyroid animals show significant up-regulation ( approximately 50% increase compared with controls) in GRK2 levels in heart and lung at 60 days after birth, whereas a 50% reduction is detected in the liver at this stage. These alterations are selective, as beta-adrenergic receptors or other G protein-coupled receptor regulatory proteins, such as G protein-coupled receptor kinase 5 or beta-arrestin-1, display a different pattern of expression changes in the hypothyroid animals. The reported changes in GRK2 levels and in the receptor/kinase ratio predict alterations in adrenergic receptor desensitization and signal transduction efficacy consistent with those observed in thyroid disorders, thus suggesting a relevant role for the modulation of GRK2 expression in this physiopathological condition.     

Expression and activity of g protein-coupled receptor kinases in differentiated thyroid carcinoma

Most of the TSH effects on the proliferation and differentiation of thyroid cells are mediated by cAMP via an adenylyl cyclase-activating Gs protein. TSH receptor responsiveness in cell cultures, is regulated by G protein-coupled receptor kinase (GRK) 2 and 5. To determine whether an alteration in activity and expression of GRKs might be associated with variable levels of TSH receptor desensitization in vivo, we studied human thyroid tissues including 21 normal tissues and 18 differentiated carcinomas. GRK activity was assayed by rhodopsin phosphorylation, and GRK protein and mRNA expressions assessed by immunoblotting and real-time quantitative RT-PCR, respectively. GRK2 and GRK5 were found as the predominant isoforms in the human thyroid. GRK5 protein expression was significantly decreased in differentiated thyroid carcinoma (P < 0.02) and paralleled a decrease in GRK mRNA expression (P < 0.02). In contrast, no difference in protein and mRNA levels of GRK2 were observed between normal and cancerous thyroid tissues. Although GRK2 protein levels correlated with GRK activities, we demonstrated a significant increase in GRK activity in differentiated thyroid carcinoma (P < 0.02). Less TSH receptor desensitization occurred in differentiated carcinoma than in normal thyroid tissue, as judged by TSH-stimulated cAMP response in human thyroid cells in primary culture. In conclusion, this study indicates that GRK2 activity and GRK5 expression have opposite regulations in cancer cells. Furthermore, the decrease in GRK5 expression may underlie the reduction in homologous desensitization of the TSH receptor in differentiated thyroid carcinoma, contributing to explain the increased cAMP levels in these tumors.     

Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases

Homologous desensitization of G protein-coupled receptors is thought to occur in several steps: binding of G protein-coupled receptor kinases (GRKs) to receptors, receptor phosphorylation, kinase dissociation, and finally binding of beta-arrestins to phosphorylated receptors. It generally is assumed that only the last step inhibits receptor signaling. Investigating the parathyroid hormone (PTH) receptor --> inositol phosphate pathway, we report here that GRKs can inhibit receptor signaling already under nonphosphorylating conditions. GRKs phosphorylated the PTH receptor in membranes and in intact cells; the order of efficacy was GRK2>GRK3>GRK5. Transient transfection of GRKs with the PTH receptor into COS-1 cells inhibited PTH-stimulated inositol phosphate generation. Such an inhibition also was seen with the kinase-negative mutant GRK2-K220R and also for a C-terminal truncation mutant of the PTH receptor that could not be phosphorylated. Several lines of evidence indicated that this phosphorylation-independent inhibition was exerted by an interaction between GRKs and receptors: (a) this inhibition was not mimicked by proteins binding to G proteins, phosducin, and GRK2 C terminus, (b) GRKs caused an agonist-dependent inhibition (= desensitization) of receptor-stimulated G protein GTPase-activity (this effect also was seen with the kinase-inactive GRK2-mutant and the phosphorylation-deficient receptor mutant), and (c) GRKs bound directly to the PTH receptor. These data suggest that signaling by the PTH receptor already is inhibited by the first step of homologous desensitization, the binding of GRKs to the receptors.     

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.     

A serine cluster prevents recycling of the V2 vasopressin receptor

Receptor recycling plays a critical role in the regulation of cellular responsiveness to environmental stimuli. Agonist-promoted phosphorylation of G protein-coupled receptors has been related to their desensitization, internalization, and sequestration. Dephosphorylation of internalized G protein-coupled receptors by cytoplasmic phosphatases has been shown to be pH-dependent, and it has been postulated to be necessary for receptors to recycle to the cell surface. The internalized V2 vasopressin receptor (V2R) expressed in HEK 293 cells is an exception to this hypothesis because it does not recycle to the plasma membrane for hours after removal of the ligand. Because this receptor is phosphorylated only by G protein-coupled receptor kinases (GRKs), the relationship between recycling and GRK-mediated phosphorylation was examined. A nonphosphorylated V2R, truncated upstream of the GRK phosphorylation sites, rapidly returned to the cell surface after removal of vasopressin. Less-drastic truncations of V2R revealed the presence of multiple phosphorylation sites and suggested a key role for a serine cluster present at the C terminus. Replacement of any one of Ser-362, Ser-363, or Ser-364 with Ala allowed quantitative recycling of full-length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the recycling S363A mutant V2Rs revealed that the recycling receptor was dephosphorylated after hormone withdrawal, whereas the wild-type V2R was not, providing molecular evidence for the hypothesis that GRK sites must be dephosphorylated prior to receptor recycling. These experiments uncovered a role for GRK phosphorylation in intracellular sorting and revealed a GRK-dependent anchoring domain that blocks V2R recycling.     

Endogenous G protein-coupled receptor kinase 6 triggers homologous beta-adrenergic receptor desensitization in primary uterine smooth muscle cells

We previously reported that G protein-coupled receptor kinase (GRK) may contribute to beta-adrenergic receptor (beta-AR) uncoupling occurring just before parturition in rat uterine muscle (myometrium). To identify the GRK involved, we set up in this study a primary cell culture retaining the morphological and functional characteristics of myometrial tissue as well as the in vivo pattern of GRK expression (GRK2, GRK5, and GRK6). In this model, homologous beta-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the beta-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A in-hibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4-6 families, we demonstrated that only the GRK4-6 family mediated beta-AR desensitization. To discriminate between endogenous GRK5 and GRK6, we attempted to inhibit their action by introducing, into myometrial cells, kinase-dead dominant-negative mutants ((K215R)GRK5 and (K215R)GRK6). Expression of (K215R)GRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of (K215R)GRK5 or (K220R)GRK2 had no effect on beta-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous beta-AR desensitization in myometrial cells.     

Functional antagonism of different G protein-coupled receptor kinases for beta-arrestin-mediated angiotensin II receptor signaling

beta-arrestins bind to G protein-coupled receptor kinase (GRK)-phosphorylated seven transmembrane receptors, desensitizing their activation of G proteins, while concurrently mediating receptor endocytosis, and some aspects of receptor signaling. We have used RNA interference to assess the roles of the four widely expressed isoforms of GRKs (GRK 2, 3, 5, and 6) in regulating beta-arrestin-mediated signaling to the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2 by the angiotensin II type 1A receptor. Angiotensin II-stimulated receptor phosphorylation, beta-arrestin recruitment, and receptor endocytosis are all mediated primarily by GRK2/3. In contrast, inhibiting GRK 5 or 6 expression abolishes beta-arrestin-mediated ERK activation, whereas lowering GRK 2 or 3 leads to an increase in this signaling. Consistent with these findings, beta-arrestin-mediated ERK activation is enhanced by overexpression of GRK 5 and 6, and reciprocally diminished by GRK 2 and 3. These findings indicate distinct functional capabilities of beta-arrestins bound to receptors phosphorylated by different classes of GRKs.     

Glucose-dependent insulinotropic polypeptide confers early phase insulin release to oral glucose in rats: demonstration by a receptor antagonist

A novel GIP receptor antagonist was developed to evaluate the acute role of glucose-dependent insulinotropic polypeptide (GIP) in the insulin response to oral glucose in rats. Antisera to an extracellular epitope of the GIP receptor (GIPR) detected immunoreactive GIPR on rat pancreatic beta-cells. Purified GIPR antibody (GIPR Ab) specifically displaced GIP binding to the receptor and blocked GIP-mediated increases in intracellular cAMP. When delivered to rats by ip injection, GIPR Ab had a half-life of approximately 4 days. Treatment with GIPR Ab (1 microg/g BW) blocked the potentiation of glucose-stimulated insulin secretion by GIP (60 pmol) but not glucagon-like peptide-1 (GLP-1, 60 pmol) in anesthetized rats. The insulin response to oral glucose was delayed in conscious unrestrained rats that were pretreated with GIPR Ab. Plasma insulin levels were approximately 35% lower at 10 min in GIPR Ab treated animals compared with controls. As a result, the glucose excursion was greater in the GIPR Ab treated group. Fasting plasma glucose levels were not altered by GIPR Ab. We conclude that release of GIP following oral glucose may act as an anticipatory signal to pancreatic beta-cells to promote rapid release of insulin for glucose disposal.     

An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.     

Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor

Signaling through beta-arrestins is a recently appreciated mechanism used by seven-transmembrane receptors. Because G protein-coupled receptor kinase (GRK) phosphorylation of such receptors is generally a prerequisite for beta-arrestin binding, we studied the roles of different GRKs in promoting beta-arrestin-mediated extracellular signal-regulated kinase (ERK) activation by a typical seven-transmembrane receptor, the Gs-coupled V2 vasopressin receptor. Gs- and beta-arrestin-mediated pathways to ERK activation could be distinguished with H89, an inhibitor of protein kinase A, and beta-arrestin 2 small interfering RNA, respectively. The roles of GRK2, -3, -5, and -6 were assessed by suppressing their expression with specific small interfering RNA sequences. By using this approach, we demonstrated that GRK2 and -3 are responsible for most of the agonist-dependent receptor phosphorylation, desensitization, and recruitment of beta-arrestins. In contrast, GRK5 and -6 mediated much less receptor phosphorylation and beta-arrestin recruitment, but yet appeared exclusively to support beta-arrestin 2-mediated ERK activation. GRK2 suppression actually increased beta-arrestin-stimulated ERK activation. These results suggest that beta-arrestin recruited in response to receptor phosphorylation by different GRKs has distinct functional potentials.     

High efficiency method for gene transfer in normal pituitary gonadotropes: adenoviral-mediated expression of G protein-coupled receptor kinase 2 suppresses luteinizing hormone secretion.

The level of LH secretion is determined by both alterations in gonadotrope responsiveness and alterations in GnRH secretion. The molecular mechanisms underlying gonadotrope responsiveness are unknown, but may include G protein-coupled receptor kinases (GRKs). Typically, GRKs phosphorylate the intracellular regions of seven-transmembrane receptors permitting beta-arrestin to bind, which prevents receptor activation of its G protein. Previously, we reported that heterologous expression of GRK2, -3, and -6 in GnRH receptor-expressing COS cells by complementary DNA transfection suppressed GnRH-stimulated inositol trisphosphate production, and that coexpression of GRK2 and beta-arrestin-2 was more inhibitory than either expressed alone. Here, we have investigated the effect of GRK2 on GnRH-stimulated LH secretion using adenovirus-mediated gene transfer in normal pituitary gonadotropes. Pituitary cells were infected with adeno-GRK2 or adeno-beta-galactosidase constructs at a multiplicity of infection of 60 (number of viral particles per cell). Seventy-two hours later, GRK2 expression was measured by enzyme-linked immunosorbent assay, and GnRH-stimulated LH secretion (10(-7) M GnRH-A for 90 min) was assayed by RIA. Adeno-beta-galactosidase infected 96-99% of the cells based on X-Gal staining. Uninfected and adeno-beta-galactosidase-infected cells exhibited endogenous GRK immunoreactivity of about 0.5 (OD405), and LH secretion of 14.8-17.7 ng/ml. Adeno-GRK2-infected cells showed a GRK2 immunoreactivity of about 2.5 (OD405) and LH secretion of 2.5 ng/ml. Therefore, adeno-GRK2 infection resulted in a 5-fold increase in the GRK2 OD405 value, which was accompanied by an 80-85% decrease in GnRH-stimulated LH secretion. GnRH-stimulated inositol trisphosphate production by gonadotropes also was inhibited, suggesting a site of action for GRK2 at phospholipase Cbeta or earlier in the signal transduction pathway. The significance of these findings is 2-fold: 1) adenoviral-mediated gene transfer permits investigation of the regulatory role of gene products in the cell of interest, the gonadotrope, rather than in heterologous cell systems; and 2) additional, stronger evidence is provided that supports a role for GRKs in setting the responsiveness of GnRH receptor signaling.     

Regulation of G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) specifically interact with the agonist-activated form of G protein-coupled receptors (GPCRs) to effect receptor phosphorylation and desensitization. Recent studies demonstrate that GRK function is a highly regulated process, and it is perhaps in this manner that a handful of GRKs (7 have been identified to date) are able to regulate the responsiveness of numerous GPCRs in a given cell type in a coordinated manner. The mechanisms by which GRK activity is regulated can be divided into 3 categories: 1) subcellular localization; 2) alterations in intrinsic kinase activity; and 3) alterations in GRK expression levels. This review will summarize our current understanding of each of these regulatory processes, and offer explanations as to how such mechanisms influence GPCR regulation under various physiologic conditions.     

The human thyrotropin receptor is predominantly internalized by beta-arrestin 2

The beta-arrestin-dependent endocytosis of the beta2-adrenergic receptor (beta2AR) has been demonstrated by confocal fluorescence microscopy. Furthermore, a constitutively activated beta2AR is also constitutively desensitized and down-regulated. To clarify the function of beta-arrestin 1 or 2 for TSH receptor (TSHR) desensitization and examine whether constitutively activated TSHR mutants are internalized in a different way, we investigated the TSHR trafficking in association with beta-arrestins in cotransfection experiments in HEK 293 cells using confocal laser-scanning microscopy. We found that both beta-arrestins are able to internalize the TSHR in HEK 293 cells. However, whereas the beta-arrestin 1-mediated TSHR internalization reached its maximum 20 min after TSH stimulation, the beta-arrestin 2-mediated TSHR internalization already reached its maximum 5 min after TSH stimulation. Furthermore, an increased basal desensitization and internalization of constitutively activated TSHR mutants N670S, S505N, and F631L cotransfected with beta-arrestin 2 could not be found. After TSH stimulation the constitutively activated mutants showed the same time course for internalization as the wild-type-TSHR. In summary, contrary to data obtained for the beta2AR, the constitutive activation of the TSHR does not influence the desensitization and time course for internalization of the receptor, and in agreement with findings for the FSH and LH receptors, these results characterize the TSH receptor as a member of the class A of G protein-coupled receptors, which have a higher affinity to beta-arrestin 2 than beta-arrestin 1 and do not colocalize with beta-arrestins in endosomes.     

β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β₂-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β₂-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.     

Functional desensitization of the extracellular calcium-sensing receptor is regulated via distinct mechanisms: role of G protein-coupled receptor kinases, protein kinase C and beta-arrestins

The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca(2+)(e)) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to beta-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and beta-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca(2+)(e)-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G alpha(q) binding (D110A) was without major effect. Overexpression of GRK 4-6 did not reduce Ca(2+)(e)-dependent inositol phosphate accumulation. Overexpression of beta-arrestin 1 or 2 revealed a modest inhibitory effect on Ca(2+)(e)-dependent inositol phosphate production (20-30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10-15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to beta-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to G alpha(q).     

G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells

We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca(2+)](i). GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca(2+)](i) in response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.