Platelet-activating factor receptor: gene expression and signal transduction

BB,

Biochi~ie~a et Biophysica Acta

ELSEVIER

Biochimica et Biophysica Acta 1259 (1995) 317-333

Review

Platelet-activating factor receptor: gene expression and signal transduction Takashi Izumi *, Takao Shimizu Department of Biochemist~', Faculo' of Medicine, Unil'ersiO'of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received 19 May 1995; revised 24 July 1995: accepted 3 August 1995

Keywords: PAF; PAF receptor; Gene; Signal transduction

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

318

2. Evidence for a cell surface PAF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Strict structural requirement and stereospecificity for the bioactivity of PAF . . . . . . . . . . . . . . . 2.2. Specific antagonists of the PAF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Specific and saturable binding of radiolabeled PAF and WEB 2086 . . . . . . . . . . . . . . . . . . . . 2.4. Driving second messenger systems through the PAF receptor . . . . . . . . . . . . . . . . . . . . . . . 2.5. Attempts to isolate the PAF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319 319 319 319 319 319

3. Molecular cloning and primary structure of PAF receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cloning of PAF receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Primary structure and properties of PAF receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Distribution of PAF-receptor mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

320 320 320 321

4. Structure and regulation of the human PAF-receptor gene . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Structure of the human PAF-receptor gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Properties of the promoter regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

321 32 I 321

5. Signal transduction via the PAF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Involvement of G-proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Phospholipid turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Inhibition of adenylate cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Protein tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Mitogen-activated protein kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. PAF-induced gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323 323 323 325 325 325 326

Abbreviations: l-acyl-2-acetyl-GPC, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine; CAT, chloramphenicol acetyltransferase; cPLA 2, cytosolic phospholipase A2; ERE, estrogen-responsive element; G-protein, guanine nucleotide regulatory protein; GRK, G-protein receptor kinase; IP 3, inositol 1,4,5-trisphosphate; LPS, lipopolysaccharide; LT, leukotriene; Lyso-PAF, 1-O-alkyl-sn-glycero-3-phosphocholine; MAPK, mitogen-activated protein kinase; PAF, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PG, prostaglandin; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PLA 2, phospholipase A 2; PLC, phospholipase C; PLD, phospholipase D; PMA, phorbol 12-myristate 13-acetate; PTK, protein tyrosine kinase; PTX, pertussis toxin; SH, Src homology; TGF, transforming growth factor; TIE, TGF-/3 inhibitory element; TNF, tumor necrosis factor; TX, thromboxane * Corresponding author. Fax: +81 3 38138732. 0005-2760/95/$09.50 © 1995 Elsevier Science B,V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 1 7 1 - 9

T. lzumi, T. Shimizu / Biochimica et Biophysica Acta 1259 (1995)317-333

318

Regulations of PAF receptor-induced signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . 1 . Characteristics of desensitization to PAF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . 2 . Involvement of protein kinases in desensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . 3 . Receptor phosphorylation by G-protein receptor kinase . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . 4 . Receptor internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . 5 . Down-regulation of PAF-receptor gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.

8.

326 326 327 327 328 328

Other features of the PAF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . I. PAF receptor-dependent internalization of PAF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . 2 . Cross-talk between the PAF receptor and other ligands . . . . . . . . . . . . . . . . . . . . . . . . . .

328

Conclusions and future prospects .

329

Acknowledgement References

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1. Introduction Platelet-activating factor (1-O-alkyl-2-acetyl-snglycero-3-phosphocholine; PAF) is a potent phospholipid mediator, causing microvascular leakage, vasodilatation, contraction of smooth muscle, and activation of neutrophils, macrophages, and eosinophils. PAF is thought to play important roles in allergic disorders, inflammations, shocks, some diseases [1-4], and also to have physiological effects on reproductive [5,6], cardiovascular [7] and central nervous systems [8]. Table 1 summarizes some of these situations. Historical stories on the discovery and identifications of PAF have been described in excellent review articles [1,2,4,9]. In early 1970's, the presence of a new factor was proposed involving histamine and serotonin release from platelets in immunized rabbits. In 1979, three independent laboratories [10-12] described the chemical structure of a new subclass of ether phospholipids that possessed biological activities identical to platelet-activating factor (PAF) and an antihypertensive polar renal lipid (APRL). The structure of PAF is shown in Fig. 1. AGEPC (acetyl glyceryl ether phosphorylcholine) and PAF-acether are also names that include structural information of this lipid, but a simple name, PAF, has found wide acceptance. Molecular heterogeneity of PAF has been observed at the sn-1 position. The 16:0 alkyl moiety is predominant, but 18:0, 17:0, and 18:1 chain species were also found. The physiological significance of the heterogeneity for the alkyl chain is unknown, though the 16:0 moiety is the most potent in biological activities. There are two synthetic routes for PAF, the remodeling and de novo pathways [4,13,14]. A variety of cells and tissues have been shown to synthesize PAF in significant quantities upon stimulation through the remodeling pathway, while some cells have the capability of producing PAF without stimulation, by the de novo synthesis. When cells are stimulated, an important intermediate, 1-O-alkylsn-glycero-3-pbosphocholine (termed lyso-PAF) is made from the PAF precursor phospholipid (1-O-alkyl-2-acyl-

328 329

329

329

sn-glycero-3-phosphocholine). This key reaction step via the remodeling pathway can occur by either a selective phospholipase (PL)A 2 that catalyzes the hydrolysis of the Table 1 Physiological and pathophysiological effects of PAF Blood cells Platelet aggregation and secretion Chemotaxis and activation of neutrophils Chemotaxis and activation of eosinophils Activation of macrophages and monocytes Stimulation of B lymphocytes Whole body Acute inflammations Allergic disorders Endotoxin shock Anaphylactic shock Disseminated intrat'ascular coagulation Central nervous system Synaptic plasticity lschemic brain damage Conuulsion Cardiovascular system Hypotension Bradycardia Negative ionotropic effect (cardiac muscle Myocardial ischemia Respiratory system Bronchoconstriction Bronchial hyperreactivity Brcmchial asthma Acute lung injury Gastrointestinal system Smooth muscle contraction of gastrointestinal tract Portal vein hypertension Glycogenolysis in the liver Peptic" ulcer Kidney Proliferation of mesangial cells Inhibition of renin release GIomerulonephritis Reproductive system Ovulation Ovoimplantation Stimulation of embryo Diseases are given in italics.

l=umi, T. Shimizu / Biochimica et Biophysica Acta 1259 (1995) 317-333

011 H2i--OmCH2--(CH2)n'-CH3 H3C~Cm

cially those isolated from herbal plants), and the chemically-synthetic compounds. The structure and potencies of various antagonists are described in review articles [2,15].

OmCH

I H2 C ~ O ~

O P~O~CH2~

i

319

CH3 CH2~ N~CH 3

\

O-

2.3. Specific and saturable binding of radiolabeled PAF and WEB 2086

CH3

n = 14-16

Fig. I. Chemical structure of PAF.

sn-2 fatty acyl residue from the PAF precursor phospholipid, or by a transacylation sequence involving a PLA 2 that metabolizes plasmalogen to lysoplasmalogen and a CoA-independent transacylase. Recent findings indicate the transacylation sequence is mainly responsible for the production of lyso-PAF [13,14]. In each case, the PLA2(s) prefers a phospholipid with arachidonate at the sn-2 position, and releases free arachidonic acid during the production of lyso-PAF. Arachidonic acid is further converted to various types of prostaglandin (PG)s, thromboxane (TX)s and leukotriene (LT)s (collectively termed eicosanoids), while lyso-PAF is converted to PAF by an acetyltransferase. Therefore, stimulation for PAF production also leads to eicosanoid production, both compounds acting synergistically. This article will discuss the characterization and regulation of the PAF receptor and PAF receptormediated intracellular signaling mechanisms.

2. Evidence for a cell surface PAF receptor

Since the discovery and structural identification of PAF, much attention has been given to its specific receptor. The following observations had suggested the existence of the cell surface receptor for PAF. 2.1. Strict structural requirement and stereospecificity .for the bioacti~'i~' of PAF An alkyl-ether bond at the sn-l position is necessary for biological activity and replacement with an ester bond leads to a loss of the activity. The acetyl moiety shows the strongest activity at the sn-2 position, and the longer acyl chain makes the less activity. At the sn-3 position, phosphorylcholine is crucial for the related activity. The naturally occurring R-chirality at the sn-2 is active, while the stereoisomer (S-form) is inactive. Phosphocholine, but not phosphoethanolamine, at the sn-3 position is also required. Such structure-activity relation is well documented in review articles [2,9]. 2.2. Specific antagonists of the PAF receptor There are three classes of PAF receptor antagonists including phospholipid analogs, natural products (espe-

More direct evidence was obtained from binding experiments, using either radiolabeled PAF or WEB 2086. The development of the radiolabeled PAF antagonist, [3H]WEB 2086, provided an excellent experimental tool for the binding assay because it is metabolically stable and nonspecific binding is low [16,17]. Specific binding sites for PAF have been reported on platelets, neutrophils, eosinophils, macrophages, monocytes, Kupffer cells, smooth muscle cells, vascular endothelial cells, tracheal epithelial cell, and various tissues such as lung, liver, brain, retina, iris, uterus etc. as discussed in a recent review [18]. 2.4. Drit:ing second messenger systems through the PAF receptor Following activation of the PAF receptor, diverse intracellular responses are elicited, including activation of PLC, PLA 2, and PLD, an increase of cytosolic Ca 2+ concentration, activation of protein kinase C (PKC), protein tyrosine phosphorylatiom and gene expression. These biochemical effects are thought to occur subsequently after the activation of the PAF receptor. The PAF-induced intracellular signals will be discussed in a later section. 2.5. Attempts to isolate the PAF receptor There are difficulties regarding the solubilization and purification of the PAF receptor. First, PAF is a phospholipid and is easily incorporated into micelles and shows high non-specific binding. The second problem is that PAF-target cells have only several hundred to thousands of PAF receptor. Furthermore there is no antagonist suitable for making an affinity column. Some attempts have been done to purify the solubilized PAF receptor using conventional column chromatographies, a PAF-human serum albumin Sepharose, or radiolabeling with a photoreactive PAF-derivative. Proteins of several different molecular masses (52, 160, 180, and 220 kDa) have been claimed to be a putative receptor for PAF as reviewed [19]. These results strongly suggest that PAF binds to a specific surface receptor in the cell membrane, but the molecular characteristics of the PAF receptor remained to be elucidated. A significant advance was made in this area when our group cloned a cDNA for a PAF receptor from guinea-pig lung [20].

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3. Molecular cloning and primary structure of PAF receptors

genomic library [25], and from an EoL-1 cell (a human eosinophilic leukemia cell line) cDNA library [26]. All human cDNAs have the same sequence in the coding region but, as discussed later, two different 5'-noncoding regions were obtained. Recently, rat [27] and mouse (lshii, S. et al., unpublished) PAF-receptor cDNAs were also cloned in our laboratory. A partial sequence of a rhesus PAF receptor was registered in GenBank (the accession No. P35366) by Behal, R.H. et al.

3.1. Cloning o f P A F receptors Xenopus laevis oocytes provide a good tool for cloning receptors and ion channels. The oocytes have a highly efficient translation machinery and are able to modify proteins post-translationally and to transport newly formed proteins to appropriate cellular compartments. The oocyte system offers another advantage in that receptors linked to G-protein-coupled PI turnover can be detected by an inward current involving a Ca2+-dependent C1 channel. Several types of receptors have been cloned using this expression system. When oocytes were injected with mRNA from guinea-pig lung, they exhibited a predominant inward current when treated with 10 -7 M PAF after a 3-day incubation. A size-fractionated cDNA library was constructed from guinea-pig lung mRNA. cRNAs from 10 pools of 30000 independent phage clones were injected into oocytes and screened by electrophysiology. Positive fractions were subfractionated by sib selection. A single clone with a 3-kbp insert was obtained [20]. Subsequently, a human PAF-receptor cDNA was isolated from leukocyte cDNA library [21,22], from an HL-60 granulocyte cDNA library [23], from a heart cDNA library [24], from a

3.2. Primao' structure and properties o f P A F receptors

The deduced amino acid sequences of PAF receptors from four species are aligned in Fig. 2. The human and guinea-pig PAF receptors have 342 amino acids, but the rat and mouse PAF receptors lack one amino acid in the third extracellular loop. The calculated molecular masses were about 39000. Hydropathy analyses indicated the presence of seven putative transmembrane domains, characteristic of a G-protein coupled receptor superfamily. The overall sequence identity among the guinea pig, human, and rat species was 74%, with 81% identity observed in the transmembrane spanning domains. Several amino acids are highly conserved compared with other G-protein coupled receptor; for example: aspartic acid (Asp-63) in the second transmembrane segment; one cysteine each in the

TMI

TM2

Human:

MEPHDSSHMDSEFRYTLFPIVYSIIF•LG•IANGYVLWVFARLYPCKKFNEIKIFMVNLTMADMLFLITLPLWIVYYQNQ

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--i~S--RV

....................

Rat:

--(~G-FRV

..............

Rhesus:

........

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GNWILPKFLCNVAGCLFFINTYCSVAFLGVITYNRFQAVTRPIKTAQANTRKRGISLSLVIWVAIVGAASYFLILDSTNT ---F

.......

-D--VH ....

L ...........................

..... F ......

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.....

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161

VPDSAGS(~VTRCFEHYEKGSVPVLIIHIFIVFSFFLVFLIILFCNLVIIRTLLMQPVQQQRNAEVKRRALWMVC~.LAV -SNK---tJI ........... K. . . . . . . C--LG--I---L ......... H---R---K ....... R. . . . . . . . . . . . . --KKD--tJI ........ P Y - - - I - W . . . . TSC . . . . . F L - F Y - - M - - - H - - - T R - - R - - - K P . . . . . . . . . . . . . . . . --N .... -~I ...................................... (208 p a r t i a l )

241

FIICFVPHHVVQLPWTLAELGFQDSKFHQAINDAHQVTLCLLSTNCVLDPVIYCFLTKKFRKHLTEKFYSMRSSRKCSRA

TM6

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.....................................

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TTDTVTEVVVPFNQIPGNSLKN

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320

-,--,---.-,--.-,,,---

G--MAI-I-HT-V-PI--

341

Fig. 2. Amino acid alignment of PAF receptors. GP, guinea pig. The Rhesus sequence is partial. One amino acid (Asp-264 in the human PAF receptor) is missing in the rat sequence. TM 1-7 indicate the putative transmembrane regions. *, two Cys residues possibly making a disulfide bond. #, Ser/Thr residues clustered at the C-terminus. Putative N-glycosylationsites are surrounded by open squares.

T. lzumi, T. Shimizu/ Biochimica et Biophysica Acta 1259 (1995) 317-333

first and the second extracellular loops (Cys-90 and Cys173) which may form a disulfide bond: and three prolines in the sixth and seventh segment (Pro-247, -254, and -290/-289). Two putative N-glycosylation sites (one near the N-terminus and the other in the second extracellular loop) were present at Asn-4 and Asn-169 in the cases of the guinea-pig and rat receptors, but Asn-4 was substituted by His in the human receptor. A cysteine in the C-terminal tail (Cys-317 or Cys-316) is also conserved and is a putative palmitoylation, membrane anchoring site. In addition, a cluster of nine Ser/Thr residues is found near the C-terminal cytoplasmic tail. Some of these residues are candidates for phosphorylation by G-protein receptor kinases (GRKs) as discussed later. Recently, a PAF acetylhydrolase in bovine brain cytosol was purified and its cDNA cloned [28,29]. The 29-kDa catalytic subunit of the enzyme has a highly homologous sequence of 29 amino acids to the N-terminal region of the PAF receptor in the 7 residues downstream from the active serine site (ser-47). So, these sequences may constitute a candidate portion for the binding to the acetyl moiety of PAF (see Scheme 1). However, a plasma acetylhydrolase showed no homology with the brain acetylhydrolase or the PAF receptor [30]. A three-dimensional computer-model for the human PAF receptor was constructed using a bacteriorhodopsin as a reference protein [31 ]. They suggested that the positively charged choline moiety of PAF is probably attracted to the negatively charged site constituted by Asp-63, Asn-285 and Asp-289 in the transmembrane domain. However, no ascertained evidence about the structure-function relationship of the PAF receptor has been obtained. Recently, a mutagenesis study of the PAF receptor showed that the phosphorylation sites of its cytoplasmic tail are involved in agonist-induced signal desensitization [32]. 3.3. Distribution

of PAF-receptor mRNA

Tissue distributions of PAF-receptor mRNA have been studied by Northern Blot analysis in guinea pig [20], rat [27,33], human cells [21], and by in situ hybridization in human peripheral lung [34]. In guinea-pig tissues, PAF-receptor mRNA is most abundant in leukocytes, followed by lung, spleen, and kidney. In rat tissues, the expression of PAF-receptor mRNA was detectable in spleen, small intestine, kidney, lung, liver, and brain, while PAF-receptor mRNA was much less abundant in thymus, stomach, pancreas, adrenal, testis, heart, and skeletal muscle. Among different portions of rat brain, PAF-receptor expression was detected in hypothalamus, meduila-pons, olfactory bulb, hippocampus, cerebral cortex, and spinal cord, but in human PAF receptor bovine PAF acetylhydrolase

321

low abundance in thalamus and cerebellum [33]. In human peripheral lung, high levels of PAF-receptor mRNA were detected in blood vessels, moderate levels in alveolar walls and peripheral airway smooth muscle [34].

4. Structure and regulation of the human PAF-receptor gene 4. I. Structure o f the human PAF-receptor gene

Studies on a human genomic library showed that the human PAF-receptor gene contains no introns in its coding region and maps to chromosome 1 [35,36]. Another study indicated that splicing of mRNA encoding PAF receptors occurs in the 5'-untranslated region between bases - 3 9 and - 38 [25]. A genomic library was screened using DNA probes corresponding to the sequences specific for the leukocyte-derived cDNA ( - 3 9 to - 1 0 4 ) [21,23] or the heart-derived cDNA ( - 39 to - 180) [24], and two distinct clones including each 5'-flanking noncoding exon (Exon 1 and Exon 2) were obtained [36]. Restriction map analysis of the region revealed that the size of the intron in the 5'-untranslated region is more than 20 kbp [35,36]. Fig. 3 shows the schematic presentation of the genomic structure for the human PAF receptor. The human PAF-receptor gene generates two different species of mRNAs (PAFR transcript 1 and PAFR transcript 2), whose expressions are driven by distinct promoters. The distributions of these two transcripts are different. PAFR transcript 1 is found ubiquitously, and most abundant in peripheral leukocytes and EoL-I cells, while the PAFR transcript 2 is detected in the heart, lung, spleen, and kidney, but not in leukocytes, EoL-I cells, or brain. The message of PAFR transcript 1 in all tissues is not due to the contamination of leukocytes in various cells, because certain cell lines (for example, JR-St cells) contain both transcripts [36]. The transcription initiation site was determined by either primer extension and 5'-RACE [36]. For the PAFR transcript 1, cytidine residues at a position - 4 0 0 (minor) and - 3 2 7 (major) were identified as start sites in EoL-I cells, and for the PAFR transcript 2, a cytidine residue at a position - 2 5 9 was identified in the heart. 4.2. Properties o f the promoter regions

The two distinct promoters are probably involved in regulating the expression of human PAF receptors in different cells and under different conditions. Their structure and function were analyzed by Mutoh et al. [36-38]. In the upstream region of Exon 1, neither TATA nor

N'I0-DSEFRYTLFPIVYSIIFVLGVIANGYVLW ::I:: :]I: :::: I :I :: :III N'45-GD_SLVQLMHQCEIWRELFSPLHALNFGIGGDSTQHVLW

Scheme~.H~m~g~ussequence~t~eenthehumenPAFrece~t~andtheb~vinePAFacety~hydr~ase.

322

T. lzumi, T. Shimizu / Biochimica et Biophysica Acta 1259 (1995) 317-333

CCAAT box is observed. The putative promoter region for Exon 1 contains consensus sequences for NF-KB and Sp-I binding as shown in Fig. 3. In addition, both the major and minor transcription start sites (at positions - 4 0 0 and - 3 2 7 ) for the PAFR transcript 1 are surrounded by a pyrimidine-rich sequence (5'-CCTCTTTCT) which is homologous to the 'Initiator' sequence (5'-CCTCATTCT). Initiator was found in the murine terminal deoxynucleotidyltransferase (TdT) gene, and is thought to act as a functional promoter related to B-cell differentiation [39,40]. During the differentiation of HL-60 cells or EoL-1 cells, increases of PAF-receptor mRNA and PAF-induced responses have been reported [21,23,26,41-43]. Consensus motifs like the Initiator may play a role in gene activation related to differentiation of blood cells. Functional analysis of the promoter region for PAFR transcript 1 using chloramphenicol acetyltransferase (CAT) gene as a reporter showed that the three sets of tandem consensus binding sites for NF-KB are important for the basal transcriptional activity and are responsible for the stimulative effects of PAF and phorbol 12-myristate 13acetate (PMA) [37]. NF-KB participates in the regulation of cytokines that play important roles in immune reaction and inflammation. Moreover, NF-KB is activated by cytokines, PMA, and lipopolysaccaride (LPS), revealing a mechanism of self-stimulatory and co-stimulatory loops [44]. One of the important roles of PAF is as a mediator of allergy and inflammation. Many inflammatory cells such as p o l y m o r p h o n u c l e a r leukocytes, m o n o c y t e s , macrophages, and lymphocytes produce PAF as well as are targets for PAF. Cytokines and LPS have been reported to increase PAF binding, PAF-induced responses, or amount of PAF-receptor mRNA; for example, LPS and tumor necrosis factor (TNF) show these effects on Mono Mac 6 cells [45,46], interferon-y on human peripheral monocytes [47], transforming growth factor (TGF)-/3 on monocyte cell lines [48], LPS on IC-12 cells [49], TNF-c~ on human PMNL [50], IL-4 and TGF-/3 on B cell lines [48,51]. PAF itself up-regulated the number of PAF binding sites two- to three-fold without affecting the affinity [51], and also stimulated PAF-receptor expression at transcriptional level

in human peripheral monocytes [52]. A recent study reported that PAF increases the precursor for NF-KB p50 mRNA in mouse intestine [53]. The consensus binding sites for NF-KB in the promoter region for PAFR transcript 1 may be involved in the expression of the PAF-receptor gene in inflammatory cells. The upstream region of the RNA start site of Exon 2 also lacks TATA and CCAAT boxes. The promoter region for the PAFR transcript 2 contains consensus sequences for AP-2, estrogen-responsive element (ERE), TGF-/3 inhibitory element (TIE), and Sp-1 [36] as shown in Fig. 3. Function of the promoter region for PAFR transcript 2 was also analyzed using CAT gene as a reporter [38]. Through the study of deletion mutants it was shown that three AP-2 sites and the Sp-I site are important for the basal transcriptional activity. It was also demonstrated that estradiol positively regulates the transcriptional activity of the CAT constructs through ERE, and TGF-/3 negatively regulates it through TIE. The ERE in the PAF gene included two half-sites of palindromic motifs with a 153 bp space. Although most responsive elements for estrogen-regulated genes have three nucleotides separating the halfpalindromes [54], widely spaced palindromic ERE motifs have been reported to work in transcriptional regulation of some genes such as ovalbumin gene [55]. PAF influences some reproductive phenomena such as ovulation [56], sperm motility [57], implantation [58], and embryonic metabolism, growth and viability [6]. Recently, the existence of a PAF receptor and its functions have been shown in a human endometrial cell line HEC-1A [5], and estrogen stimulates expression of PAF-receptor gene in human endometrial cells (Sato, S., unpublished data). Estrogen may regulate the expression of PAF-receptor gene and control PAF effects on the reproductive systems. TGF-/3 seems to control the gene expression of the PAF receptor in diverse ways. TGF-/3 decreased the transcriptional activity through TIE located in the promoter region of PAFR transcript 2 [38], while it was reported to increase the mRNA for the PAF receptor in monocytes and B cell lines [48]. TGF-/3 may increase PAFR transcript 1 (not PAFR transcript 2), which mainly presents in monocytes and B cell lines.

promoter region 5'-noncodingregion codingregion

-900~"¢~'~

~[327

5~mX

Exon 1

-39 1 ATG

1020 TAG

I I

Exon 2

Fig. 3. Genomicstructure of the human PAF receptor.ERE, estrogen-responsiveelement; TIE, TGF-/3 inhibitoryelement. 'Initiator" is a homologous sequenceto the 'Initiator' of the murine terminal deoxynucleotidyltransferasegene presentedin Ref. [39]. See text for details.

T. Izumi, T. Shimizu / Biochimica et Biophysica Acta 1259 (1995) 317-333

cellular effector systems through more than one G-protein [21,63,64].

5. Signal transduction via the PAF receptor

PAF activates phospholipid turnover through PLC, PLA 2, phosphatidylinositol 3-kinase (PI3K), and PLD. PAF also activates many kinases, e.g., PKC, mitogenactivated protein kinase (MAPK), GRK, and protein tyrosine kinase (PTK)s. Some of these signal transduction mechanisms are related to the expression of specific genes. These PAF-induced signals are coupled to individual cellular responses. The research area of ligand-activated intracellular signals is now expanding, and many signal transduction mechanisms not only typical for G-protein coupled receptors but also for growth factor receptors can be involved in the signals induced by the PAF receptor. Fig. 4 shows an overview of the PAF receptor-coupled signal transduction pathways. 5.1. Involvement o f G-proteins

Many studies have indicated that PAF-induced signals involve guanine nucleotide regulatory proteins (G-proteins) as reviewed [18,19,59]. The binding of PAF to membranes stimulates GTPase activity [60,61]. GTP and its stable analogues caused shifts in the binding of PAF [61,62]. The type of G-proteins involved in the PAF responses may differ from cell to cell and also depend on effector systems because some processes coupled with G-proteins are sensitive to pertussis toxin (PTX) treatment while others are resistant. Molecular cloning of PAF-receptor cDNAs clearly showed that PAF receptors belong to a G-protein coupled receptor superfamily. The cloned PAF receptor expressed in Xenopus oocytes, COS-7 cells, CHO cells, and RBL-2H3 cells were also linked functionally to

Extracellular space

5.2. Phospholipid turnover 5.2.1. Phospholipase C

PAF activates polyphosphoinositide turnover in many systems including platelets, macrophages, monocytes, neutrophils, eosinophils, B cell lines, smooth muscle cells, endothelial cells, hepatocytes, Kupffer cells, keratinocytes, glomerular mesangial cells, astrocytes, and so on as reviewed [18,65]. This pathway is mediated by PLC, and two products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, are generated. IP3 mobilizes the intracellular Ca 2÷ and diacylglycerol activates PKC. The increase of IP3 occurred within 5-10 s of PAF challenge, and appears to be independent of extracellular Ca 2+. However, the PAF-induced increase in cellular Ca 2+ is predominantly due to influx of extracellular Ca-"+ rather than IP3-dependent mobilization of intracellular Ca" + [18,65]. The mechanism of this influx remains to be elucidated. In the G-protein family, Gi, Gq, G o, Gp (Gll, G14, Gls, and GI6), Gz, an unknown G-protein has been proposed to activate PLC, and among them, G~, G o and G z are PTXsensitive [66]. Several studies have used PTX as a tool to monitor the involvement of G-proteins in PLC activation. PTX causes ADP-ribosylation of the o~ subunit of G-protein and inactivates it. The most well-established pathway for activation of PLCt~ is through the subunits of PTX-insensitive Gq, GI1, G14, and GI6 proteins [67,68]. Also, the /3y subunit has recently been shown to activate PLCt~ and this is thought to be the mechanism for the PTX-sensitive activation of PLC [68,69]. Previous reports indicate the

PAF-R

1

AA, lys

PG, TX, LT PAF

323

~ DAG

"

~

/

C~a2+

~

Fig. 4. Intracellularsignalingevents coupledto the PAF receptor.See text for abbreviations.

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involvement of G-protein in PAF-coupled PLC activation, but the type of PLC activated by PAF has not been identified. In macrophages and neutrophils the polyphosphoinositide turnover induced by PAF was sensitive to PTX [70,71], while in platelets it was only partially sensitive [72]; U937 cells were insensitive to PTX [73]. In some systems, PTX blocked PAF-induced polyphosphoinositide turnover but did not affect the Ca 2÷ rise [74,75]. This may be because that small amount of IP3 produced by PAF is sufficient to elicit maximum Ca 2+ mobilization as speculated before [76]. In the cloned PAF receptor expressed in CHO cells, PAF-induced IP3 production was insensitive to PTX [63] but in the one expressed in RBL-2H3 cells it was partially sensitive [64]. These data also indicate that PAF activates PLC through coupling to both PTX-sensitive and PTX-insensitive G-proteins and this could vary from cell to cell. Another mechanism for PAF-induced PLC activation is through its phosphorylation by PTKs [68]. Many receptors (e.g., those for epidermal growth factor, PDGF, nerve growth factor) which have intrinsic tyrosine kinase activity are involved in the activation of PLCr. These receptors cause phosphorylation of a tyrosine residue (Tyr-783) of PLCr~ that is essential for its activation [77]. Generally, tyrosine kinase-activated PLC~(s) increase(s) Ca 2+ more slowly and for longer duration in contrast to a transient increase in Ca 2+ by PLCt~ [68]. G-protein coupled receptors that have no intrinsic tyrosine kinase activity can also activate PLCr, though the precise mechanism has not been elucidated. Involvement of PTKs in PAF-induced activation of PLCr has been proposed in platelets and A431 cells [72,78]. Recent work clearly showed that PAF induces the tyrosine phosphorylation and activation of PLCr in a human B cell line [79,80]. 5.2.2. Phospholipase A 2 In many cells arachidonic acid is released from membrane phospholipids in response to receptor-mediated signals, and cytosolic PLA 2 (cPLA 2) is thought to be responsible for this hydrolysis. The released arachidonic acid is further metabolized to biologically active eicosanoids, such as PGs, TXs, and LTs [81-83]. In addition, in the remodeling pathway for PAF synthesis, lyso-PAF and arachidonic acid are released simultaneously by the action of PLA2(s) with a preference for phospholipids containing arachidonate at sn-2 position [4,13,14]. PAF stimulates release of arachidonic acid and eicosanoids in many cells and tissues such as platelets, neutrophils, eosinophils, macrophages, smooth muscle cells, epithelial cells, heart, and lung, as reviewed [18]. PAF-induced eicosanoid production is thought to play an important role in the following processes; coronary vasoconstriction and cardiac contractility via production of TXA 2 and LTC 4 [84], contraction of mesangial cells via PGE 2 [85], bronchoconstriction via LTs and TXA 2 produced by eosinophils [86], liver hemodynamics via TXA 2

[87], lethal shock in the mouse via LTs [88], and aggregation and activation of neutrophils via lipoxygenase products [89,90]. In the guinea-pig atrium, PAF activates a K ÷ channel by production of an arachidonic acid metabolite, possibly LTC 4 [91]. Recently 5-1ipoxygenase-deficient mice have been developed by two research groups, and these mice became resistant to the lethal effects of shock induced by PAF, indicating that LTs may play important downstream roles in PAF-induced shock, as 5-1ipoxygenase is a key enzyme for LT production [92,93]. An agonist-responsive cPLA 2 has been purified and cloned [94,95]. The regulation of this cPLA 2 by Ca 2÷ and MAPK is well established and involves membrane translocation and phosphorylation, respectively [96]. Although the mechanism for cPLA 2 stimulation by G-proteins remains uncertain, this may not involve direct coupling between a pure G-protein subunit and cPLA 2 [97]. The role of MAPK in PAF-induced PLA 2 activation will be discussed later in this paper. Previous studies have demonstrated that PAF activates PLA 2 through a PKC-dependent mechanism [98100] and that it is regulated by intracellular cyclic AMP levels [101,102]. It remains unclear whether these effects of PKC or cyclic AMP are due to their regulation of MAPK or not. 5.2.3. Phosphatidylinositol 3-kinase PI3K is a phospholipid kinase that carries out 3-phosphorylation of phosphatidylinositol and has received much attention recently because its main product, phosphatidylinositol (3,4,5)-trisphosphate, is a potential second messenger involved in membrane ruffling, superoxide generation in neutrophils, and glucose transport control in adipocytes [103-105]. Phosphatidylinositol (3,4,5)-trisphosphate activates an isoform of a typical PKC, PKC; [106]. The PI3K pathway has been well described in signaling for growth factors, but the activity of this kinase is also controlled by heterotrimeric G-proteins [103]. The mechanism(s) for the activation of PI3K can vary. One mechanism involves is the tyrosine phosphorylation of the regulatory p85 subunit, another is a coupling between PI3K and some other tyrosine-phosphorylated molecules [107]. However, recently Ras, in its activated GTP-form, was reported to associate directly with PI3K and activate it [108], and furthermore, a new form of PI3K controlled by /3y subunit of G-protein was documented [109]. PAF has been reported to stimulate PI3K in human neutrophils [110] and in a human B cell line [80]. In the B cell line, tyrosine phosphorylation of the p85 subunit has been demonstrated [110], but other control mechanisms cannot be excluded. The biological roles of PI3K in PAF signaling have not been elucidated. 5.2.4. Phospholipase D PLD catalyzes the hydrolysis of phosphatidylcholine in response to a variety of stimuli, playing an important role in signal transduction of cells by yielding phosphatidic

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acid which is further metabolized to diacylglycerol through the action of phosphatidate phosphohydrolase or to lysophosphatidic acid and free fatty acid (e.g., arachidonic acid) by PLA 2 [97,111 ]. Phosphatidic acid and its metabolites may act as signal transduction molecules in various cell responses. PAF has been shown to stimulate PLD in human and rabbit neutrophils [112,113], promonocytic U937 cells [114], mouse peritoneal macrophages [115], human erythroleukemia cells [116], A431 cells [117], human endometrium [118], and rat renal mesangial cells [119]. Recently, we observed PAF-induced activation of PLD in CHO cells expressing the cloned PAF receptor [1201. The signal transduction pathway that activates PLD has not been fully defined, but regulatory roles of Ca 2+, PKC, PTK, small GTP-binding proteins, and ADP-ribosylation factor (ARF) have been demonstrated [97]. In PAF-induced PLD activation, Ca 2+ [ 113,120], PKC [ 114,119,120], and PTK [120] are thought to be involved. Recently, one form of PLD has been purified from pig lung [121]. The latter provides new avenues of study of the PLD pathway in receptor-mediated signal transduction. 5.3. Inhibition o f adenylate cyclase

PAF has an inhibitory effect on adenylate cyclase activation stimulated by many agents such as forskolin and PGI 2 in platelets [122,123], isoproterenol in bovine pulmonary arterial endothelial cells [124], and TSH and forskolin in porcine thyroid cells [125], and PGE 2 and salbutamol (a /3-adrenoceptor agonist) in guinea-pig alveolar macrophages [101]. In CHO cells expressing the cloned PAF receptor, PAF inhibited forskolin-stimulated cAMP accumulation and this effect was completely abolished by PTX treatment [63]. These data indicate that the PAF receptor is coupled with PTX-sensitive G-protein, Gi. However, the physiological role of the inhibitory effects of PAF on adenylate cyclase has not been clarified. On the contrary, PAF increased the cAMP level in macrophagelike cells P388D~ indirectly through the formation of PGE~ [126]. 5.4. Protein ~rosine kinases

PTKs were originally thought to play an important role in the signal pathways mediated by growth factors receptors, but recent studies indicate that they are also involved in G-protein mediated signal transduction [127]. Previous studies using anti-phosphotyrosine antibody showed that PAF induces tyrosine phosphorylation of numerous cellular proteins in rabbit platelets [72,128], human neutrophils [129], rat liver Kupffer cells [100], and human B cell lines [79,130]. PAF-induced protein phosphorylation has been also observed in CHO cells expressing the cloned PAF receptor [63,120]. These responses were rapid, and were blocked by PAF receptor antagonists [72,128,! 31]. Some

325

of the phosphorylated bands have been identified as follows: pp60 ..... in rabbit and human platelets [128,131] and A431 cells [78], PLC~,j in rabbit platelets [132] and a human B cell line [80], p85 regulatory subunit of PI3K in a human B cell line [80], Fyn and Lyn (both src-related PTKs) in a human B cell line [80], and MAPK in CHO cells expressing the cloned PAF receptor [63,120]. The Src homology (SH)2 and SH3 domains have been shown to play key roles in tyrosine kinase pathways. Proteins that contain SH2 domains interact with phosphotyrosinecontaining proteins and become tyrosine phosphorylated and activated [133]. Proteins such as pp60 c-~c, Fyn, Lyn, PLC:, l, and p85 regulatory subunit of PI3K that are tyrosine-phosphorylated by PAF-stimulation contain both SH2 and SH3 domains. Although the molecular mechanisms through which the PAF receptor activates tyrosine kinase signals have not been clarified, the tyrosine-phosphorylation of src family proteins may trigger the activation of PLC~, and PI3K [80,134]. Actually, PTK inhibitors (e.g., genistein, erbstatin, etc.) have been demonstrated to inhibit PAF-stimulated PLC activity in platelets [72,135,136], neutrophils [137], a human B cell line [79], and A431 cells [781. PTK inhibitors also inhibited PLD in human neutrophils [112]. A phosphotyrosine phosphatase inhibitor, sodium vanadate, stimulated PGE 2 production in rat Kupffer cells [138], while PTK inhibitors diminished PAF-induced eicosanoids production in murine peritoneal macrophages [139] and rabbit platelets [135], indicating that PTK cascades may also be involved in PLA: activation. Recently, PTK inhibitors were shown to inhibit the PAF-induced activation of PLD and MAPK in CHO cells expressing the cloned PAF receptor [ 120]. These data indicate that a PTK pathway may also be involved in the PAF-induced PLD, MAPK and PLA 2 activation in addition to PLC and PI3K activation. 5.5. Mitogen-actiL~ated protein kinase

Tyrosine-phosphorylated MAPK was observed after PAF stimulation in platelets [140], human B cell lines [130], guinea-pig neutrophils [141], and CHO cells expressing the cloned PAF receptor [63,120]. MAPKs are considered to play a key role in the kinase cascade that translocates signals from the membrane to the nucleus [142-144]. Many stimuli that trigger cell differentiation and cell-cycle transition activate MAPKs. MAPKs transmit their signals by phosphorylating downstream components such as transcription factors, c-jun, c-myc, and p62 Tcv. Another important feature of MAPK is that this kinase phosphorylates Ser-505 of cPLA 2, and that this phosphorylation is unequivocal for the complete activation of the enzyme together with Ca 2+ mobilization [96]. MAPKs are activated also through heterotrimeric G-protein-mediated mechanisms [ 145-147]. In addition to its actions as a proinflammatory mediator,

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PAF causes proliferative and differentiating signals in non-inflammatory ceils. PAF induces neurite outgrowth in PC I2 pheochromocytoma cells [148] and up-regulates the proliferation of lymphocytes [149]. PAF also stimulates gene expression including the expression of primary response genes such as c-los, c-jun, and TIS-I in many cells (discussed later). These primary response genes appear to play important roles in PAF-induced cell growth and differentiation. Some of these effects may be explained by the PAF-induced MAPK activation. It is well established that Raf-1 plays a central role in the regulation of MAPK in eukaryotes [150]. In the signal transduction leading from tyrosine kinase-related receptors such as growth hormone receptors, G r b 2 / S O S / R a s system activates Raf [151] through 14-3-3 [152,153]. Raf phosphorylates and activates MAPK kinase (also termed MEK), leading to MAPK stimulation. Rhodopsin type receptors coupled to heterotrimeric G-proteins can activate the MAPK pathway. Activated c~i (a PTX-sensitive G-protein subunit) and /3y-complexes stimulate Raf activity in a Ras-dependent manner, while activated ~Xq (a PTX-insensitive G-protein subunit) and /3y-complexes may stimulate Raf by a PKC-dependent mechanism [150], It has been reported that MAPK kinase and MAPK activation are mediated both by G~ and by PTX-insensitive G-proteins [ 145,147,154,155]. Furthermore, PTX-sensitive and -insensitive pathways may be involved in PAF-induced MAPK activation in CHO cells expressing the cloned PAF receptor [63]. The other study using the cloned PAF receptor showed that the PAF receptor activates MAPK through the Ca 2÷-, PKC-, and PTK-dependent pathway [120]. In guinea-pig neutrophils, PAF activates MAPK through two distinct pathways, one Ca2+-dependent, and the other sensitive to wortmannin (a PI3K inhibitor) [141]. Thus, there might be more than one pathway for the PAF-induced activation of MAPK that remains to be further elucidated.

5.6. PAF-induced gene expression PAF has been reported to induce rapid and transient expression of early response genes in many cells and tissues in a receptor-mediated manner as shown in Table 2. The induction of the expression of these genes indicates that PAF possesses proliferative and differentiating effects

Table 2 PAF-induced expression of early response genes Cell and tissues

Gene

Human monocytes Human neuroblastoma cells Human B cell lines A-431 cells Rabbit corneal epithelial cells Rat astroglial cells Rat hippocampus

c-fos c-los, c:fos c-los, c-los, c:fos, c-los,

Reference

c-jun TIS-1 c-jun, zif/268 zif/268

Ho et al. [227] Squinto et al. [148] Mazer et al. [177] Tripathi et al. [228] Bazan et al. [229] Dell' et al. [230] Marcheselli et al. [231]

in many cells as has been shown for neuroblastoma cells [148] and lymphocytes [149]. PAF-induced expression of genes other than early primary response genes has been reported; a CR1 (a C3b receptor) gene in neutrophils [156], a IL-3 gene in human umbilical cord blood mononuclear cells [157], IL-I, IL-6, TNF, and IL-2Ra genes in monocytes, an IL-6 gene in neutrophils, a TNF gene in NK cells, an IL-6 gene in endothelial cells as reviewed [158]. PAF itself has been reported to increase expression of the PAF-receptor gene as described before [37,48,51]. These induced genes may play a role in PAF-mediated cell injury, inflammation, and immune responses, but the mechanism remains undetermined. There exist several lines of data indicating the presence of intracellular PAF receptor(s) and their involvement in gene induction [ 159,160], while ascertained molecular evidence has yet to be obtained. Regulation of gene expression is a complex phenomenon. The involvement of Ca R+ influx and a cyclic AMP/Ca2+-response element [159], PKC and PTKs [19] in PAF-induced gene expression has been discussed in previous reviews. The precise mechanisms of PAF-induced gene expression have not been elucidated, but the activation of the MAPK pathway may explain the proliferative effects and the gene-regulatory effects of PAF as described before in this article.

6. Regulations of PAF receptor-induced signals 6.1. Characteristics of desensitization to PAF The action of PAF has been reported to show homologous desensitization to repeated or long standing application of PAF in the following processes; secretion of vasoactive amines from rabbit platelets [161], contraction of guinea-pig ileal smooth muscle [162], exocytosis of azurophilic and lysosomal granules in human neutrophils [163], aggregation of rabbit platelets [164], contraction of guinea-pig myometrium [165], effiux of 45Ca2+ from preloaded cultured bovine aortic endothelial cells [166], rat paw edema [167], dopamine release in PC-12 cell line [ 168], and so on. Many PAF-induced cellular responses are also desensitized; e.g., GTPase activity [169], IP3 production [170], Ca 2+ mobilization [171,172], and protein phosphorylation [173]. The cloned PAF receptors expressed on Xenopus oocytes, CHO cells and COS cells also show such a desensitization of IP3 production and Ca 2+ mobilization [20-22,32]. Studies on heterologous desensitization have been cartied out. In thrombin-treated platelets PAF-induced protein phosphorylation was desensitized, but PAF treatment did not influence the protein phosphorylation resulting from thrombin treatment [173]. In cultured LLC-PKI cell line, PAF- or ONOll113 (a T X A 2 analogue)-challenged cells showed heterologous desensitization to the other agonists

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[174]. The heterologous desensitization of PAF-induced Ca 2+ influx was also observed in cells pretreated with bradykinin or to a less extent with ATP, and conversely, pretreatment of cells with PAF affected only partially the Ca2+-response elicited by bradykinin or ATP in neuroblastoma × glioma hybrid, NG 108-15 cells [175]. PAF heterologously desensitizes U46619 (a TXA 2 agonist)-evoked PLC activation in human platelets, but had no such effects on rat vascular smooth muscle cells [176]. No heterologous desensitization in Ca 2÷ mobilization was observed in B cell lines between PAF and anti-lgM antibody [177], in neurohybrid NG 108-15 cells between PAF and bradykinin, endothelin, angiotensin II, or ATP [178], and in neurohybrid NCB-20 cells between PAF and bradykinin or ATP [179]. PAF-induced activation of GTPase and Ca 2+ mobilization are inhibited by pretreatment of leukocytes with fMLP, C5a and interleukin-8 [180]. Consequently, it appears as if heterologous desensitization between the same set of agents detected in some cells are not observed in others. These data indicate that receptor cross-desensitization is regulated by several mechanisms with selectivity for different types of stimulants and may differ from cell to cell. Desensitization of receptors may occur by uncoupling the transducer system (e.g., G-proteins) from the receptor and also by ligand-induced receptor internalization. Protein kinases are thought to be main participants in both desensitization mechanisms. Down-regulation of IP3 receptor is another suggested explanation for heterologous desensitization [181 ].

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cient for PAF-induced desensitization since pretreatment with PMA inhibited the Ca 2+ responses and staurosporine attenuated subsequent desensitization to PAF although without completely abolishing it [188]. These studies indicate the involvement of PKC in modulation of PAF-induced signals although modulation of PKC alone cannot explain the entire mechanism that accounts for PAF desensitization. In a recent study, the epitope-tagged human PAF receptor expressed in a rat basophilic cell line (RBL-2H3 cells) was phosphorylated by PAF and also by PMA and dibutyryl cyclic AMP [64]. Staurosporine caused complete inhibition of PAF receptor phosphorylation by PMA but only partial inhibition of that elicited by PAF. Receptor phosphorylation by PAF and PMA (but not by dibutyryl cyclic AMP) correlated with desensitization as measured by a decrease in both PAF-stimulated GTPase activity in membranes and cellular Ca 2+ mobilization. Thus, the PAF receptor can be phosphorylated by three kinds of kinases (PKA and PKC and other kinases), while phosphorylation by PKC and receptor kinase(s) may be involved in desensitization to PAF [64]. PAF has been reported to activate PKCs and translocate them from cytosol to membrane in alveolar macrophages [189], murine endothelioma cell lines [190], cerebral microvessels [191], and human platelets [192]. The PAF-induced activation and membrane translocation of PKCs may be one of the mechanisms for desensitization.

6.2. Inuoluement o f protein kinases in desensitization

6.3. Receptor phospho~lation by G-protein receptor kinase

Although the molecular mechanism of PAF-induced desensitization remains to be elucidated, in some systems PKC is thought to be involved in these phenomena. If the membranes were isolated after preincubation of endothelial cells from bovine pulmonary artery with PAF or PMA, the adenylate cyclase activity was decreased by 70 and 90%, respectively, with lowered affinity for isoproterenol, indicating that interaction of PAF with these cells leads to a /3-adrenergic receptor desensitization probably through a phosphorylation mechanism involving PKC [124]. Pretreatment with PKC activators (e.g., PMA, teleocidin, aplysiatoxin, or mezerein) resulted in desensitization of PAF signals in many cells, including endothelial cells from bovine pulmonary artery [124], rat liver cells (C-9 cell line) [182], human bone marrow derived macrophages [183], human platelets [184], guinea-pig peritoneal eosinophils [185], and rabbit neutrophils [186]. On the other hand, in rabbit platelets, staurosporine potentiates PAF-stimulated PLC activity but does not block desensitization by PAF, indicating that PLC activity is negatively affected by PKC while other mechanism(s) than PKC activation is involved in PAF desensitization [187]. Another study on CHRF-288-11 megakaryocytic cells showed that PKC activation appears to be necessary but not suffi-

The desensitization mechanisms for G-protein-coupled receptors have been studied in most detail with /32-adrenergic receptors. PKA and GRK has proven to be responsible for desensitization by phosphorylating Ser/Thr residues in the carboxyl-terminal tail of /3-adrenergic receptor [193,194], However, in comparison with G~-coupled receptors, little is known about the desensitization mechanisms for receptors coupled to PLC [195]. Recent work indicates that some PLC-coupled receptors (e.g., m3 muscarinic receptors [196], formylpeptide and Csa receptors [197], a j-adrenergic receptors [198], and cholecystokinin receptors [199]) are as well phosphorylated in an agonist-dependent manner, and it has been shown that the substance P receptor is a good substrate for /3-adrenergic receptor kinases 1 and 2 (new terms, GRK2 and GRK3) [200]. The GRK family plays an important role in the signal shut-down of PLC-coupled receptors as well as G~-coupled receptors. In human leukocytes, PAF induced translocation of a GRK from cytosol to membrane and stimulated its activity to a larger extent than did isoproterenol [201]. Since agonist-induced GRK translocation is considered to be the first step in GRK-mediated homologous desensitization [202], GRK possibly plays a role in the shut-down of PAF signals. Using mutant receptors expressed in CHO cells,

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we analyzed the role of the cytoplasmic tail [32]. A truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of IP3 and intracellular Ca 2+ concentrations. Similar findings were noted in another mutant, in which the distal Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors potently activated other signals (MAPK kinase, arachidonic acid release, and inhibition of adenylate cyclase) in a greater extent than did the wild type receptor. Thus, while the carboxyl-terminai cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does play a critical role in signal attenuation induced by the agonist through phosphate acceptors. It was also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was phosphorylated by the recombinant GRK2, suggesting that GRK or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization [32]. On the other hand, the involvement of PKC is not prominent in the CHO cells [32]. Thus, GRK is a candidate kinase for rapid and homologous desensitization of the PAF receptor, and second messenger kinases such as PKC and PKA may provide feedback regulatory loops and heterologous desensitization of the PAF receptor. 6.4. Receptor internalization

As a consequence of agonist exposure, many Gprotein-coupled receptors undergo rapid internalization (sequestration). However, little is known about the internalization of the PAF receptor. Loss of PAF binding sites [169,203] and decreased affinity for high affinity sites for PAF [204,205] was reported in desensitized platelets. Whether these data indicate either receptor internalization, uncoupling of receptor-effector systems, or modification in binding properties of the receptor has not been ascertained. A recent study [206] showed that PMA caused a complete loss of PAF receptors in human neutrophils from the cell surface as determined by specific radioligand binding, and that disruption of PMA-treated neutrophils exposed latent PAF binding sites. Furthermore, cytochalasin B, an agent capable of disrupting microfilaments, attenuated PMA-induced loss of PAF binding sites. It was thought that the cellular basis for PMA-induced PAF receptor loss from human neutrophils likely involves internalization or cellular redistribution of the PAF receptor [206]. Whether such mechanisms occur in PAF-stimulated neutrophils or in other cells has not been clarified. Another recent study showed that incubation of COS-7 cells with PAF resulted in the disappearance of approximately 20% of the epitopetagged PAF receptor expressed on the cell surface [207]. Some amino acid residues highly conserved in many members of the G-protein-coupled receptor, such as Tyr326 in the distal 7th transmembrane domain of the human /32-adrenergic receptor [208], Arg-139 in the proximal second intracellular loop and Ala-263 in the distal third intracellular loop of gastrin-releasing peptide receptor

[209], have been reported to be involved in receptor internalization. The corresponding amino acid residues are also conserved in all cloned PAF receptors (Arg-115, Ala-230, and Tyr-293 in the human PAF receptor), but whether or not these amino acids are involved in PAF-receptor internalization remains to be clarified. 6.5. Down-regulation of PAF-receptor gene expression

Desensitization is a rapid process occurring within a few minutes after agonist exposure, while another regulation mechanism, down-regulation of receptor gene expression is seen in a half or one hour after agonist exposure and lasts for several hours [18,210]. In human peripheral monocytes, it was reported that a transient elevation of cAMP induced by PGE 2, cholera toxin, or forskolin inhibited PAF-receptor expression, and dibutyryl cAMP also reduced the expression of PAF receptors without changing the stability of PAF-receptor mRNA. Furthermore, the inhibition of PAF-receptor mRNA accumulation was associated with diminished responsiveness to PAF and decreased PAF receptor protein expression on the cell surface [211]. Such a cAMP-dependent mechanism for PAF receptor down-regulation was also observed in hepatic Kupffer cells [102]. A recent report demonstrated that prolonged exposure of human promonocytic U937 cells to carbarmyl-PAF, a non-metabolizable analogue of PAF, reduced the numbers of PAF receptors by 50-75%, paralleled with a decreased expression level of the PAF-receptor gene [210]. This report indicates that a long-term exposure of PAF may down-regulate the gene expression of PAF receptor. On the contrary, other studies have shown that PAF up-regulates PAF binding sites [51] and stimulates PAF-receptor expression [52] as discussed in an earlier section. Most G-protein-coupled receptors have a conserved cysteine in the C-terminal cytoplasmic tail near the seventh transmembrane spanning region. This cysteine is known to be palmitoylated in rhodopsin [212], the /32-adrenergic receptor [213], and the a2A-adrenergic receptor [214]. For the /32-adrenergic receptor, this cysteine was shown to be important for Gs-coupling and agonist-promoted desensitization [215], while for the a2A-adrenergic receptor this cysteine plays a critical role in down-regulation of receptor number after prolonged agonist exposure [216]. Cys-317 or -316 is conserved in the C-terminal cytoplasmic tail in all cloned PAF receptors. Whether this cysteine is palmitoylated and if so, what function the palmitoylation has in the PAF-promoted signal remains to be clarified. 7. Other features of the PAF receptor 7.1. PAF receptor-dependent internalization of PAF

Cells have a huge capacity to take up and metabolize PAF, and this process may proceed by receptor-independent endocytosis, receptor-dependent endocytosis, or

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mechanisms other than endocytosis as discussed [217]. Incorporated PAF initially localizes to the plasma membrane outer leaflet which can be extracted with albumin, but it rapidly becomes albumin insensitive, yet remains associated with plasma membrane [218,219]. Then, PAF might be deacetylated and acylated in the plasma membrane, and move to Golgi/granule storage sites in acylated forms as reported [218]. PAF receptors were not thought to directly participate in incorporation processes but could stimulate them by activating the ability of translocating PAF across the plasmalemma [219,220]. However, a recent study showed a mechanism for PAF receptor-dependent internalization of PAF in COS-7 cells expressing the cloned human PAF receptor [207]. The molecular mechanism of this internalization remains to be elucidated and whether or not the PAF receptors are involved in ligand internalization in cells other than COS-7 cells should be clarified. 7.2. Cross-talk between the P A F receptor and other ligands

Some cell types (e.g., mast cell, basophil, endothelial cell) produced predominantly l-acyi-2-acetyl-sn-glycero3-phosphocholine (l-acyl-2-acetyl-GPC) rather than PAF in response to stimuli [221-223]. The effects of l-acyl-2acetyl-GPC on human neutrophils were examined [223,224]. l-Acyl-2-acetyl-GPC induced a rapid increase in cytosolic Ca 2+ in the neutrophils although the potency of 1-acyl-2-acetyl-GPC was 300-fold lower than that of PAF [224]. The dose response curves for both l-acyl-2acetyl-GPC and PAF were shifted in a parallel fashion by a PAF-receptor antagonist, L-652,731. Furthermore, preincubation of neutrophils with l-acyl-2-acetyl-GPC, caused a dose-dependent inhibition of /3-glucuronidase and lysozyme release induced by a subsequent stimulation with PAF [224]. I-Acyl-2-acetyl-GPC acted as a stimulus for LTC 4 release with similar kinetics to those of PAF and these effects were inhibited by a PAF-receptor antagonist, WEB 2086, with the same IC50 value [223]. These data suggested that l-acyl-2-acetyl-GPC may represent, under certain circumstances, a modulator of PAF effects via mechanisms shared with PAF, though no direct interaction between the PAF receptor and 1-acyl-2-acetyl-GPC has been shown. A cross-talk between LPS and the PAF receptor has been suggested [225,226]. It was shown that in platelets, the LPS-induced Ca 2÷ increase and aggregation was blocked by PAF-receptor antagonists, and that in Xenopus oocytes and CHO cells, LPS induced Ca 2+ increase only when PAF receptors were expressed, and furthermore, specific PAF binding was displaced and reversibly dissociated by LPS.

8. Conclusions and future prospects PAF plays a crucial role in inflammation and immune systems [1-4]. It is also involved in proliferation and

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differentiation of various cells including neuronal and blood cells [148,149]. Since molecular cloning of the PAF receptor in 1991 [20], progress has been made for elucidation of signal transduction, and gene expression of the receptor. This review summarizes these advances made in various laboratories including ours. The following problems still remain: (1) in vivo roles of PAF and its receptor in physiological systems (central nervous, cardiovascular, respiratory, renal etc.) (2) role of PAF in various disorders, and potential benefit of PAF antagonists in these diseases; (3) relationship of PAF with other signal molecules such as interleukins, or cell adhesion molecules; (4) structuralfunction relationships of the PAF receptor, including the amino acid sequence for the binding site of PAF to its receptor and mechanism of signal shut-down (uncoupling, receptor internalization and down-regulation; (5) identification of another type of PAF receptors, and putative intracellular PAF receptor, (6) nature and structure of PAF-synthesizing enzymes; and (7) the detailed understanding of the regulation of PAF synthesis and its cellular release. Some of these areas will be addressed by establishing transgenic/knock-out mice of the PAF receptor, an ongoing project in our laboratory.

Acknowledgements We are grateful to I. Ferby for pertinent comments. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare of Japan, and by grants from the Uehara Memorial Foundation, Human Science Foundation, the Yamada Science Foundation, and the Ono Medical Research Foundation.

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