©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Pathway Desensitizes Chemotactic Receptor-induced Calcium Signaling via Inositol Trisphosphate Receptor Down-regulation (*)

(Received for publication, November 3, 1994)

Zen-ichiro Honda (1)(§) Tomoko Takano (2) Naoto Hirose (1) Takeshi Suzuki (1) Akira Muto (3) Shoen Kume (3) Katsuhiko Mikoshiba (3) Kohji Itoh (1) Takao Shimizu (2)

From the  (1)Department of Internal Medicine and Physical Therapy, and (2)Department of Biochemistry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan and the (3)Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Desensitization of a chemotactic receptor is an adaptive process that terminates inflammation. Although homologous desensitization can be well explained by the action of specific receptor kinases, the mechanisms of heterologous desensitization remain elusive. As an approach to evaluate the roles of G(q) pathway in desensitization of calcium signaling, we expressed a constitutively active G(q)alpha mutant (G(q)alpha Q-L) together with platelet-activating factor (PAF) receptor in Xenopus laevis oocytes. G(q)alpha Q-L expression completely attenuated the calcium-sensitive chloride current and the Ca release elicited by PAF. The G(q)-mediated desensitization could not be ascribed to G protein/receptor uncoupling via receptor phosphorylation, because (i) PAF-induced inositol 1,4,5-trisphosphate (IP(3)) synthesis was only partially suppressed and (ii) a mutated PAF receptor devoid of all Ser and Thr in the third cytoplasmic loop and in the C-terminal tail was also completely desensitized by G(q)alpha Q-L. In G(q)alpha Q-L expressing oocytes, microinjection of IP(3) failed to elicit the calcium response, and the IP(3) receptor, detected by a specific antibody, disappeared. Thus, the G(q)-mediated desensitization can be most likely explained by IP(3) receptor down-regulation. These novel mechanisms may explain in part heterologous desensitization in chemotactic factor-stimulated inflammatory cells.


INTRODUCTION

Cloning of chemotactic factor receptor cDNAs had broadened understanding of activation-desensitization processes in agonist-stimulated inflammatory cells. Platelet-activating factor (PAF)(^1)(1, 2) , C5a(3) , and thromboxane A(2)(4) receptors were found to possess the seven-transmembranous structure characteristic of G protein-coupled receptors. Subsequent studies revealed that these receptors could transduce calcium signaling via newly emerged pertussis toxin-insensitive G protein alpha subunits, Galpha and Galpha(5, 6, 7, 8, 9, 10) .

Once a G protein-coupled receptor is activated with the first stimulus, cells often become unresponsive to the same agonist or even to others. Homologous and heterologous desensitizations are fundamental adaptive processes that terminate inflammatory reactions. A widely accepted mechanism of the homologous desensitization is a phosphorylation of an agonist-occupied receptor by specific receptor kinases, such as beta-adrenergic receptor kinases(11, 12) , and subsequent receptor/G protein uncoupling. We recently obtained evidence that phosphorylation of Ser and Thr residues in the C-terminal cytoplasmic tail by beta-adrenergic receptor kinase-like kinases plays crucial roles in the homologous desensitization of the cloned PAF receptor(13) . A recent study using an artificial yeast system revealed that a pheromone receptor, which coupled to tripartite G proteins, causes a homologous desensitization even in cells devoid of G proteins(14) . These observations suggest that signals downstream of G proteins may not be indispensable for homologous desensitization in some systems.

In contrast to homologous desensitization, heterologous desensitization occurs in a previously unoccupied receptor (or in its signaling pathway) and rationally requires signals downstream of the primarily activated G proteins. Thus, to better understand heterologous desensitization, it is crucial to know if G protein activation itself will induce desensitization. In an attempt to address this question and to elucidate the underlying mechanisms, we expressed wild and mutated PAF receptors together with a constitutively active G(q)alpha mutant(15, 16) . We used a gene expression system in Xenopus oocytes, since this system allows us (i) to co-express multiple genes in a single cell, (ii) to sensitively detect calcium response by electrophysiological methods, and (iii) to microinject second messengers. We now report evidence that G(q) activation attenuates subsequent chemotactic receptor-mediated calcium signaling and that the main event related to this desensitization is down-regulation of the inositol 1,4,5-trisphosphate (IP(3)) receptor.


MATERIALS AND METHODS

Plasmids

To prepare a mutant PAF receptor (PAFR-mut) lacking possible phosphorylation sites, C-terminal 40 amino acids of guinea pig PAF receptor cDNA were deleted, and Thr-212 was substituted with Ala (see Fig. 1B). A constitutively active guinea pig G(q)alpha mutant in which Gln-209 was substituted with Leu (G(q)alpha Q-L), were prepared as described(15, 16) . These plasmids were subcloned into pBluescript SK(-) (Toyobo Inc. Tokyo).


Figure 1: PAF-induced membrane current was inhibited by the co-expression of G(q)alpha Q-L but not by G(q)alpha. A, typical recordings of PAF-induced calcium-sensitive chloride current. Oocytes injected with cRNAs in combination (25 ng of each cRNA/oocyte) were voltage-clamped, and the membrane currents elicited by the superfusion of 10 nM PAF (denoted by horizontalbars) were assayed. Oocytes injected with PAFR cRNA (control) or with PAFR + G(q)alpha cRNAs (+Galpha) displayed typical biphasic responses, whereas those injected with PAFR + G(q)alpha Q-L cRNAs (+Galpha Q-L) failed to respond to PAF. B, schematic transmembrane structures of PAFR and PAFR-mut, and PAF-induced response in oocytes. (The C-terminal side from the fifth membrane-spanning domain is represented. In the C-terminal cytoplasmic tail, 4 Ser (S) and 5 Thr (T) residues are present.) The bar graph summarizes the 10 nM PAF-induced membrane currents. Oocytes expressing PAFR or PAFR + G(q)alpha responded to PAF (640 ± 200 nA, n = 6, and 700 ± 150 nA, n = 6, respectively; mean ± S.E.), whereas co-expression of G(q)alpha Q-L suppressed the response (5 ± 10 nA, n = 7; mean ± S.E.). Oocytes injected with PAFR-mut + G(q)alpha cRNAs responded to PAF (540 ± 130 nA, n = 4; mean ± SE), but those injected with PAFR-mut + G(q)alpha Q-L did not (0 ± 0 nA, n = 4; mean ± SE). C, oocytes were injected with 25 ng/oocyte of PAFR cRNA and various amounts of G(q)alpha Q-L cRNA, and 10 nM PAF-induced response was examined. G(q)alpha Q-L cRNA dose-dependently suppressed the current. The vertical bars denote S.E. (n = 4-6).



Gene Expression in Xenopus laevis Oocytes

Stage VI-VII oocytes were treated with collagenase (type II, Sigma) and manually defoliculated as described previously(1) . Plasmids were linearized with appropriate restriction enzymes located downstream of the coding regions, and cRNA was synthesized in vitro as described previously(1, 2) . Oocytes were injected with cRNAs, incubated for 48 h in modified Bath's saline (15 mM Tris-HCl, pH 7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO(3), 0.3 mM Ca(NO(3))(2), 0.4 mM CaCl(2), 0.8 mM MgSO(4)) at 22 °C and subjected to functional assays.

Western Blotting of Xenopus IP(3) Receptor

Crude oocyte membrane was prepared exactly as described(17) . Crude membrane proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and Xenopus IP(3) receptor was probed with a specific rabbit antibody generated against C-terminal 155 amino acids of a cloned Xenopus IP(3) receptor(17) . The first antibodies were detected with peroxidase-conjugated anti-rabbit IgG, and, for color development, we used 3,3`-diaminobenzidine tetrahydrochloride and hydrogen peroxide.

Functional Assays of Oocytes

Detection of the calcium-dependent chloride current was done as described(1, 2) . In brief, oocytes were voltage-clamped at -80 mV in a bath perfused with oocyte saline (5 mM HEPES-NaOH, pH 7.4, 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl(2)) supplemented with 1 mg/ml bovine serum albumin (solution A) and challenged with superfusion of PAF or with an intracellular injection of IP(3) lithium salt (Boehringer Mannheim) or with calcium chloride. Membrane current was continuously monitored, and occasionally, the reversal potential was measured (around -20 mV) to confirm that the observed current was indeed the chloride current.

Ca efflux from oocytes was assayed essentially as described previously(18) . 20 oocytes were labeled with Ca (the final concentration, 1.85 MBq/ml) in 500 µl of calcium-free solution A for 3 h at room temperature and then were thoroughly washed with solution A and incubated in 500 µl of the same medium. 500 µl of the medium was collected before and after the addition of PAF, at defined intervals, and replaced with 500 µl of fresh solution A. Radioactivity in the medium collected at each interval was counted.

Measurement of IP(3) content in oocytes was performed as follows. 10 oocytes in 200 µl of solution A were stimulated with PAF for the indicated time, and the reaction was quenched by the addition of 70 µl of 20% perchloric acid followed by vigorous vortexing. The sample were centrifuged at 10,000 times g for 5 min at 4 °C, and then the supernatant was neutralized with 10 M KOH. Precipitated salt was again removed by centrifugation, and the supernatant was examined for IP(3) content by a competitive radioreceptor assay using the D-myo-inositol 1,4,5-trisphosphate assay system (Amersham Corp.).


RESULTS AND DISCUSSION

The present study was designed to address two interrelated questions. First, are downstream events of G(q) activation sufficient to desensitize the chemoattractant-induced calcium signaling? And, second, if G(q) activation attenuates subsequent calcium signaling, what is the mechanism? As an approach to answer the questions, we expressed a GTPase-deficient mutant of guinea pig G(q)alpha (G(q)alpha Q-L) (15, 16) that has been demonstrated to activate downstream signals, including phospholipase C and MAP kinases(19) . (^2)As a model of a potential target of G(q)-dependent heterologous desensitization, we used the guinea pig PAF receptor (PAFR) and its mutant (PAFR-mut). In PAFR-mut, Ser/Thr residues in the C-terminal tail, which play a crucial role in homologous desensitization(13) , were totally removed by deletion. In addition, a single Thr in the 3rd cytoplasmic loop was substituted with Ala (see the schematic membrane structures in Fig. 1B). cRNAs of these constructs were prepared in vitro and injected in combination (25 ng of each cRNA/oocyte, if not otherwise stated) into Xenopus oocytes.

We first examined the PAF (10 nM) induced calcium response by monitoring the calcium-operated chloride current(1, 2) . As shown in Fig. 1, in oocytes expressing PAFR alone, there was a considerable response to 10 nM PAF, and co-expression of G(q)alpha Q-L with PAFR completely suppressed this response. Co-expression of native G(q)alpha was without effect (Fig. 1). Viability of the oocytes, as checked by resting membrane potential, was not affected by G(q)alpha Q-L expression (-40 ± 12 mV versus -38 ± 15 mV, in G(q)alpha and G(q)alpha Q-L expressing oocytes, respectively; mean ± S.D., n = 4). The G(q)alpha Q-L-induced loss of PAF sensitivity could not be ascribed to failure of PAFR expression, since PAF-induced IP(3) synthesis was only partially suppressed in G(q)alpha Q-L expressing oocytes (see below and Fig. 2B). As shown in Fig. 1C, the effect depended on the amount of microinjected G(q)alpha Q-L cRNA. G(q)alpha Q-L also desensitized PAFR-mut; the PAFR-mut-mediated membrane current completely disappeared in G(q)alpha Q-L-expressing cells but not in G(q)alpha-expressing cells (Fig. 1B). Thus, in contrast to desensitization by the homoligand(13) , the G(q)-mediated desensitization could not be fully explained by the phosphorylation of Ser/Thr in the 3rd cytoplasmic loop and in the C-terminal tail.


Figure 2: G(q)alpha Q-L expression abolished PAF-induced Ca efflux but only partially inhibited IP(3) synthesis. A, 20 oocytes injected with PAFR+G(q)alpha (opencircle) or PAFR + G(q)alpha Q-L (closedcircle) cRNAs (25 ng of each cRNA/oocyte) were labeled with Ca, and Ca efflux elicited by 10 nM PAF (horizontalbar) was measured. Co-expression of G(q)alpha Q-L suppressed Ca efflux. These results were a representative of two independent examinations. B, 10 oocytes injected with PAFR+G(q)alpha (opencolumns) or with PAFR + G(q)alpha Q-L (closedcolumns) cRNAs (25 ng of each/oocyte) were challenged with 10 nM PAF, and IP(3) accumulation was measured. Co-expression of G(q)alpha Q-L partially inhibited IP(3) synthesis by 40% at 1 min and by 26% at 5 min. The verticalbars denote S.E., n = 3. These results were a representative of three independent experiments.



To analyze the machinery needed for the G(q)-mediated desensitization, we dissected the signaling pathway by measuring changes in key second messengers, calcium and IP(3). Oocytes were labeled with Ca, and the PAF-induced efflux of radioactivity was followed. PAF elicited a rapid and significant Ca efflux from oocytes injected with PAFR and G(q)alpha cRNAs. In the oocytes expressing G(q)alpha Q-L together with PAFR, this response disappeared, (Fig. 2A), thereby indicating that G(q)alpha Q-L-suppressed elevation of intracellular calcium.

We next measured change in the IP(3) level by IP(3)-specific radioreceptor assay (Fig. 2B). Basal IP(3) levels were below the detection limit in oocytes injected with G(q)alpha Q-L/PAFR cRNAs as well as those injected with G(q)alpha/PAFR cRNAs (detection limit; 0.25 pmol/10 oocytes), probably reflecting the rapid metabolism of IP(3)(20) . In the oocytes expressing G(q)alpha together with PAFR, PAF elicited a time-dependent increase in IP(3) level. In contrast to complete loss of the calcium efflux and the calcium-dependent chloride current, PAFR-mediated IP(3) elevation was only partially inhibited by the co-expression of G(q)alpha Q-L, by 40% at 1 min, and by 26% at 5 min. These observations led to the notion that prolonged activation of G(q)alpha attenuated the PAFR-mediated calcium response by modifying the IP(3)-induced calcium release, which is mediated by IP(3)-sensitive calcium channel (tetrameric complex of IP(3) receptor)(21) .

To determine whether or not the IP(3)-induced calcium release was suppressed in oocytes expressing active G(q)alpha, we microinjected IP(3) into oocytes under the recording of the calcium-dependent chloride current. As shown in Fig. 3, microinjection of 40 pmol of IP(3) elicited a large inward current in oocytes expressing G(q)alpha, whereas the response was almost completely suppressed in G(q)alpha Q-L-expressing oocytes. As G(q)alpha Q-L did not suppress the current directly activated by microinjection of 50 pmol CaCl(2) (Fig. 3A), the unresponsiveness to IP(3) was not likely to be due to altered channel functions.


Figure 3: G(q)alpha Q-L suppressed IP(3)-induced calcium response, and down-regulated IP(3) receptor. A, oocytes injected with G(q)alpha or G(q)alpha Q-L cRNA (25 ng/oocyte) were microinjected with 40 pmol of IP(3) or with 50 pmol of CaCl(2) under condition of recording of the membrane current. IP(3) elicited a distinct inward current in oocytes injected with G(q)alpha cRNA but not in those injected with G(q)alpha Q-L cRNA. CaCl(2) elicited almost comparable responses in the both groups of oocytes. B, the bargraph summarizes IP(3)-induced membrane currents. The amplitudes of the currents induced by 40 pmol of IP(3) were 960 ± 150 nA in G(q)alpha-expressing cells and 20 ± 40 nA in G(q)alpha Q-L-expressing cells (mean ± S.E., n = 6). C, crude membrane fraction of oocytes was subjected to Western blotting using anti-Xenopus IP(3) receptor antibody. In Exp.1, 50 ng of G(q)alpha or G(q)alpha Q-L cRNA was injected, and 40 µg of crude membrane was subjected to Western blotting. IP(3) receptor was detected in G(q)alpha-expressing oocytes, whereas the immunoreactivity disappeared in G(q)alpha Q-L expressing oocytes. In Exp. 2, G(q)alpha Q-L cRNA was diluted (1:10, 5 ng/oocyte; 1:100, 0.5 ng/oocyte), and injected. Water, or G(q)alpha cRNA (50 ng/oocyte) injected oocytes were used as controls. 20 µg of the membrane fraction was used. G(q)alpha Q-L cRNA dose dependently down-regulated the IP(3) receptor; G(q)alpha Q-L cRNA more than 5 ng/oocyte abolished IP(3) receptor-immunoreactivity.



We next asked whether the loss of IP(3) responsiveness is accompanied by change in the level of IP(3) receptor molecule. In control oocytes injected with G(q)alpha cRNA, a single immunoreactive protein that corresponds to Xenopus IP(3) receptor (17) was detected by a specific polyclonal antibody raised against the cloned Xenopus IP(3) receptor (17) (Fig. 3C, Exp.1). In G(q)alpha Q-L expressing cells, this imunoreactivity almost completely disappeared (Fig. 3C, Exp.1). Decrease in the immunoreactivity depended on the amount of G(q)alpha Q-L cRNA; oocytes injected with G(q)alpha Q-L cRNA at 0.5 ng/oocyte contained an almost comparable immunoreactivity to that in oocytes injected with water or with G(q)alpha cRNA (control), whereas the immunoreactive band disappeared in oocytes injected with G(q)alpha Q-L cRNA at more than 5 ng/oocyte (Fig. 3C, Exp.2).

These model experiments using a heterologous expression system revealed two novel findings concerning desensitization mechanisms of chemoattractant receptors. First, the continuous activation of downstream signals of G(q) was sufficient to desensitize subsequent chemoattractant-induced calcium signaling. Second, one of the major mechanisms of the G(q)-induced desensitization was down-regulation of the IP(3) receptor molecule. The second point readily explains the heterologous desensitization of calcium signaling. Recently Quian et al.(22) found that expression of a GTPase-deficient Galpha, a member of G(q)alpha family, desensitized both bombesin- and PDGF-stimulated calcium mobilizations in Swiss3T3 cells. Since IP(3) receptor is the first convergent point of the calcium signaling pathways originated from these different classes of receptors, the observed IP(3) receptor desensitization/down-regulation may explain the heterologous desensitization in Swiss3T3 cells.

Heretofore, several mechanisms for heterologous desensitization of G protein-coupled receptors have been proposed, including receptor phosphorylation by signal-dependent kinases, such as protein kinase C (20) , and down-regulation of G proteins(23) . These scenarios, which affect receptor-mediated phospholipase C activation, could not fully explain the G(q)-dependent desensitization. However, these mechanisms may explain the observed partial inhibition of IP(3) production. This point should be studied further.

Recently, IP(3) receptor down-regulation was noted in rat cerebellar granular cells and in SH-SY5Y human neuroblastoma cells continuously exposed to acetylcholine(24, 25) . Using an active G(q)alpha mutant, we obtained evidence that the down-regulation is not a ligand-specific phenomenon but that rather activation of G(q) pathway is sufficient for the down-regulation. The loss of IP(3) sensitivity due to the down-regulation was found to be the main reason for the G(q)-induced heterologous desensitization of the PAF receptor-originated calcium signaling. These observations also imply that chemotactic factor receptors coupling to the G(q)-family potentially elicit the same desensitization mechanisms. The next important problem is to determine if the proposed mechanisms can explain impaired leukocyte functions in inflammatory or autoimmune disorders(26, 27, 28) . The precise mechanisms of down-regulation of the IP(3) receptor remain unclear. Elevation of intracellular levels of IP(3) and calcium ion appear to be required for both the reversible inactivation of the channel function (29) and the degradation (25) of IP(3) receptor/calcium channel. In an in vitro study, IP(3) receptor was found to be a good substrate for a calcium-activable neutral protease, calpain(30) . However, the role of calpain in vivo is still controversial(25) . The Xenopus oocyte system, in which these second messengers or a calpain-specific inhibitor, calpastatin (31) can be readily introduced intracellularly, may serve as a useful system for studying the IP(3) receptor turn-over.


FOOTNOTES

*
This work was supported in part by grants-in-aid for specific research on priority areas, Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan and grants from the Japan Health Science Foundation, the Uehara Memorial Foundation, and the Yamanouchi Foundation for Research on Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel: 81-3-3815-5411; Fax: 81-3-3815-5954.

(^1)
The abbreviations used are: PAF, platelet-activating factor; IP(3), inositol 1,4,5-trisphosphate; Galpha, alpha-subunit of G protein; PAFR, PAF receptor.

(^2)
T. Watanabe, I. Waga, I., Z. Honda, K. Kurokawa, and T. Shimizu, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. H. Okano (Department of Molecular Neurobiology, University of Tsukuba) for invaluable discussions, H. Ichijo for excellent technical assistance, and M. Ohara for helpful comments.


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