Activation-induced subcellular redistribution of G alpha(s) is dependent upon its unique N-terminus

The heterotrimeric G protein subunit, alpha(s), can move reversibly from plasma membranes to cytoplasm in response to activation by GPCRs or activating mutations. We examined the importance of the unique N-terminus of alpha(s) in this translocation in cultured cells. alpha(s) contains a single site for palmitoylation in its N-terminus, and this was replaced by different plasma membrane targeting motifs. These N-terminal alpha(s) mutants were targeted properly to plasma membranes, capable of coupling activated GPCRs to effectors, and able to constitutively stimulate cAMP production when they also contained an activating mutation. However, when activated by a constitutively activating mutation or by agonist-activated beta-AR, these N-terminal alpha(s) mutants failed, for the most part, to undergo redistribution from plasma membranes to cytoplasm, as assayed by immunofluorescence microscopy, or from a particulate to soluble fraction, as assayed by subcellular fractionation. These results highlight the importance of the extreme N-terminus of alpha(s) and its single site of palmitoylation for facilitating activation-induced translocation and provide insight into the mechanism of this G protein trafficking event.     

Differential regulation of corticotropin releasing factor 1alpha receptor endocytosis and trafficking by beta-arrestins and Rab GTPases

The corticotropin releasing factor (CRF) type 1alpha receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1alpha receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1alpha receptor activation leads to the selective recruitment of beta-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1alpha receptor and beta-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, beta-arrestin2-green fluorescent protein (GFP) co-localized with CRF1alpha receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, beta-arrestin2 and CRF1alpha receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that beta-arrestin2 association with CRF1alpha receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1alpha receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1alpha receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that beta-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1alpha receptor resensitization is regulated in an atypical manner by Rab GTPases.     

Visualization of agonist-induced association and trafficking of green fluorescent protein-tagged forms of both beta-arrestin-1 and the thyrotropin-releasing hormone receptor-1.

A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.     

Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and beta-arrestin 2 during homologous desensitization of the substance P receptor

The substance P receptor (SPR) is a G protein-coupled receptor (GPCR) that plays a key role in pain regulation. The SPR desensitizes in the continued presence of agonist, presumably via mechanisms that implicate G protein-coupled receptor kinases (GRKs) and beta-arrestins. The temporal relationship of these proposed biochemical events has never been established for any GPCR other than rhodopsin beyond the resolution provided by biochemical assays. We investigate the real-time activation and desensitization of the human SPR in live HEK293 cells using green fluorescent protein conjugates of protein kinase C, GRK2, and beta-arrestin 2. The translocation of protein kinase C betaII-green fluorescent protein to and from the plasma membrane in response to substance P indicates that the human SPR becomes activated within seconds of agonist exposure, and the response desensitizes within 30 s. This desensitization process coincides with a redistribution of GRK2 from the cytosol to the plasma membrane, followed by a robust redistribution of beta-arrestin 2 and a profound change in cell morphology that occurs after 1 min of SPR stimulation. These data establish a role for GRKs and beta-arrestins in homologous desensitization of the SPR and provide the first visual and temporal resolution of the sequence of events underlying homologous desensitization of a GPCR in living cells.     

Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo

G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.     

Long-term agonist stimulation of IP prostanoid receptor depletes the cognate G(s)alpha protein in membrane domains but does not change the receptor level

Iloprost (IP) stimulation (1 microM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous G(s)alpha protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, G(i)alpha and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-G(s)alpha fusion protein. The endogenous G(s)alpha decreased, but the level of Flag-hIPR-G(s)alpha protein did not change. The specific depletion of domain-bound pool of G(s)alpha as consequence of iloprost stimulation was also demonstrated in membrane domains prepared according to alkaline treatment plus sonication protocol (detergent-free procedure of Song et al.). Our data further indicated that in control, quiescent cells only a very small amount of IP prostanoid receptor was present in DIMs together with large amount of its cognate G(s)alpha protein. Expressed in quantitative terms, DIMs contained 30-40% of the total cellular amount of G proteins whereas the content of IP prostanoid receptors was 1-3%. The dominant portion (>95%) of FhIPR as well as FhIPR-G(s)alpha was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-G(s)alpha distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the alpha subunit of Na,K-ATPase, transmembrane form of CD58 and CD44). All these proteins are known to be fully solubilised by detergent and thus unable to float in density gradient. Our data indicate that (i) long-term agonist stimulation of IP prostanoid receptor is associated with preferential decrease of its cognate G protein G(s)alpha from membrane domains; receptor level is unchanged. (ii) Very small fraction (1-3%) of total cellular amount of receptors is recovered in DIMs together with roughly 40% of G proteins. These data suggest a "supra-stoichiometric" arrangement of G proteins and corresponding receptors in DIMs.     

The adenosine A2A receptor interacts with the actin-binding protein alpha-actinin

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist. Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.     

Extra-long Gαs variant XLαs protein escapes activation-induced subcellular redistribution and is able to provide sustained signaling

Murine models indicate that Gαs and its extra-long variant XLαs, both of which are derived from GNAS, markedly differ regarding their cellular actions, but these differences are unknown. Here we investigated activation-induced trafficking of Gαs and XLαs, using immunofluorescence microscopy, cell fractionation, and total internal reflection fluorescence microscopy. In transfected cells, XLαs remained localized to the plasma membrane, whereas Gαs redistributed to the cytosol after activation by GTPase-inhibiting mutations, cholera toxin treatment, or G protein-coupled receptor agonists (isoproterenol or parathyroid hormone (PTH)(1-34)). Cholera toxin treatment or agonist (isoproterenol or pituitary adenylate cyclase activating peptide-27) stimulation of PC12 cells expressing Gαs and XLαs endogenously led to an increased abundance of Gαs, but not XLαs, in the soluble fraction. Mutational analyses revealed two conserved cysteines and the highly charged domain as being critically involved in the plasma membrane anchoring of XLαs. The cAMP response induced by M-PTH(1-14), a parathyroid hormone analog, terminated quickly in HEK293 cells stably expressing the type 1 PTH/PTH-related peptide receptor, whereas the response remained maximal for at least 6 min in cells that co-expressed the PTH receptor and XLαs. Although isoproterenol-induced cAMP response was not prolonged by XLαs expression, a GTPase-deficient XLαs mutant found in certain tumors and patients with fibrous dysplasia of bone and McCune-Albright syndrome generated more basal cAMP accumulation in HEK293 cells and caused more severe impairment of osteoblastic differentiation of MC3T3-E1 cells than the cognate Gαs mutant (gsp oncogene). Thus, activated XLαs and Gαs traffic differently, and this may form the basis for the differences in their cellular actions.     

G-protein-coupled receptor-associated A-kinase anchoring proteins AKAP5 and AKAP12: differential trafficking and distribution

A-kinase Anchoring Proteins (AKAPs) define an expanding group of scaffold proteins that display a signature binding site for the RI/RII subunit of protein kinase A. AKAP5 and AKAP12 are multivalent (with respect to protein kinases and phosphatases) and display the ability to associate with the prototypic member of G protein-coupled receptors, the beta(2)-adrenergic receptor. We probed the relative abundance, subcellular distribution and localization of AKAP5 and AKAP12 in human embryonic kidney HEK293 and epidermoid carcinoma A431 cells. HEK293 cells are relatively rich in AKAP5 (found mostly in association with the cell membrane); whereas A431 cells are rich in AKAP12 (found distributed both in the cytoplasm and in association with the cell membrane). In biochemical analysis of subcellular fractions and in whole-cell imaging, the membrane localization of AKAP5 was decreased in response to treating cells with the beta-adrenergic agonist isoproterenol, whereas membrane association of AKAP12 was increased initially in response to agonist treatment. These data demonstrate quantitatively a clearly different pattern of AKAP-receptor association for AKAP5 versus AKAP12. AKAP5 remains associated with its G-protein-coupled receptor, at the cell membrane, docked with the receptor during agonist-induced internalization and later receptor recycling after agonist wash-out. AKAP12-receptor docking, in contrast, is dynamic, driven by agonist stimulation (accounting for movement of AKAP12 from the cytoplasm to the cell membrane). AKAP12 then is internalized with the beta(2)-adrenergic receptor, but segregates away from the G-protein-coupled receptor upon recycling of the internalized receptor to the cell membrane. Thus these homologous, AKAPs that dock G-protein-coupled receptors have markedly different patterns of trafficking, docking, and re-distribution.     

A dominant-negative strategy for studying roles of G proteins in vivo

G proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the alpha subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg2+-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein alpha subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of Galphai, Galphaq, or Galphas. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M2 muscarinic receptor activated K+ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K+ channels (GIRK) into HEK 293 cells that stably express the M2 muscarinic receptor. The presence of the Galphai carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Galphai minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Galpha carboxyl peptide in random order, pcDNA-GalphaiR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Galphai minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Galphaq or Galphas had no effect on M2 muscarinic receptor stimulation of the K+ channel.     

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.     

Agonists activate Gi1 alpha or Gi2 alpha fused to the human mu opioid receptor differently

As preferential coupling of opioid receptor to various inhibitory Galpha subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1 alpha or C352I Gi2 alpha in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35 S]GTPgammaS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR-C351I Gi1 a exhibited an activation profile similar to the non-fused receptor while hMOR-C352I Gi2 alpha was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin-stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1 alpha and Gi2 alpha mainly. In contrast both fusion constructs were able to initiate ERK-MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1 alpha than Gi2 alpha and that the coupling efficacy is clearly agonist-dependent.     

Spatiotemporal interactions of myristoylated alanine-rich C kinase substrate (MARCKS) protein with the actin cytoskeleton and exocytosis of oxytocin upon prostaglandin F2alpha stimulation of bovine luteal cells

In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F2alpha (PGF2alpha) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF2alpha treatment and 2) evaluate PGF2alpha-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF2alpha (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 microM). Treatment of cells expressing wild-type MARCKS-GFP with PGF2alpha and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF2alpha treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.     

Endocytosis as a Biological Response in Receptor Pharmacology: Evaluation by Fluorescence Microscopy

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method –the Q-Endosomes algorithm– that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT_2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.     

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.     

Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N-Terminal Truncation of mGluR1α

The metabotropic glutamate receptor mGluR1alpha in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide-bonded dimer on sodium dodecyl sulphate-polyacrylamide gels. Dimerization of mGluR1alpha takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N-terminal domain of mGluR1alpha, truncated just before the first transmembrane domain (NT-mGluR1alpha), we show that the N-terminal domain is secreted as a soluble disulphide-bonded dimeric protein. In addition, the truncated N-terminal domain can form heterodimers with mGluR1alpha when both proteins are cotransfected into HEK 293 cells. However, mGluR1alpha and its splice variant mGluR1beta did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N-terminal domain of mGluR1alpha is sufficient for dimer formation, other domains in the molecule must regulate the process.     

Selective resistance to parathyroid hormone caused by a novel uncoupling mutation in the carboxyl terminus of G alpha(s). A cause of pseudohypoparathyroidism type Ib

G(s) is a heterotrimeric (alpha, beta, and gamma chains) G protein that couples heptahelical plasma membrane receptors to stimulation of adenylyl cyclase. Inactivation of one GNAS1 gene allele encoding the alpha chain of G(s) (G alpha(s)) causes pseudohypoparathyroidism type Ia. Affected subjects have resistance to parathyroid hormone (PTH) and other hormones that activate adenylyl cyclase plus somatic features termed Albright hereditary osteodystrophy. By contrast, subjects with pseudohypoparathyroidism type Ib have hormone resistance that is limited to PTH and lack Albright hereditary osteodystrophy. The molecular basis for pseudohypoparathyroidism type Ib is unknown. We analyzed the GNAS1 gene for mutations using polymerase chain reaction to amplify genomic DNA from three brothers with pseudohypoparathyroidism type Ib. We identified a novel heterozygous 3-base pair deletion causing loss of isoleucine 382 in the three affected boys and their clinically unaffected mother and maternal grandfather. This mutation was absent in other family members and 15 additional unrelated subjects with pseudohypoparathyroidism type Ib. To characterize the signaling properties of the mutant G alpha(s), we used site-directed mutagenesis to introduce the isoleucine 382 deletion into a wild type G alpha(s) cDNA, transfected HEK293 cells with either wild type or mutant G alpha(s) cDNA, plus cDNAs encoding heptahelical receptors for PTH, thyrotropic hormone, or luteinizing hormone, and we measured cAMP production in response to hormone stimulation. The mutant G alpha(s) protein was unable to interact with the receptor for PTH but showed normal coupling to the other coexpressed heptahelical receptors. These results provide evidence of selective uncoupling of the mutant G alpha(s) from PTH receptors and explain PTH-specific hormone resistance in these three brothers with pseudohypoparathyroidism type Ib. The absence of PTH resistance in the mother and maternal grandfather who carry the same mutation is consistent with current models of paternal imprinting of the GNAS1 gene.     

Agonist-induced tyrosine phosphorylation of Gq/G11 alpha requires the intact structure of membrane domains

Stimulation of receptors coupled to G(q)/G(11) protein may induce phosphorylation on a tyrosine residue of the alpha subunit of this G protein, which is an essential event for G(q)/G(11) activation. Here we observed that in HEK293 cells stably expressing high levels of thyrotropin-releasing hormone (TRH) receptors and G(11)alpha protein the maximal tyrosine phosphorylation of G(q)/G(11)alpha was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G(q)/G(11)alpha protein levels did not change during this treatment. Incubation of intact cells with beta-cyclodextrin (beta CD) for 40 min prior to hormone exposure significantly decreased the rapid transient tyrosine phosphorylation. Subsequent replenishment of cholesterol levels reversed the former negative effect of beta CD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in beta CD-treated cells. These data indicate that the preserved integrity of plasma membrane domains/caveolae is required for complete agonist-induced phosphorylation of G(q)/G(11)alpha.     

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.