Platelet-derived growth factor receptors and phospholipase C activation

Kidney International, Vol. 41(1992), pp. 575—580

Platelet-derived growth factor receptors and phospholipase C activation THOMAS 0. DANIEL and DANA A. KUMJIAN Nephrology Division, Department of Medicine, and Department of Cell Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

Platelet-derived growth factor (PDGF) has been assigned a cal signals. Both a and /3 receptor types are membrane spanning broad range of biological roles in contexts ranging from devel- glycoproteins having extracellular immunoglobulin-like doopment and oncogenesis to wound healing and atherogenesis mains which participate in ligand binding, a single membrane [1]. Originally assayed as mitogenic activity released from spanning domain, and an intracellular tyrosine kinase domain platelets, it is now appreciated that, in addition to its prolifer- homologous to the functional kinase domain of the c-src proative actions, PDGF stimulates other processes including tooncogene and its related non-receptor tyrosine kinases [5]. chemoattraction, deposition of extracellular matrix and neu- This kinase domain is interrupted in both PDGF a and /3 ronotropism. All these biological activies are mediated through receptors by an "insert" domain, the presence of which defines transmembrane cell surface tyrosine kinase receptors. a subclass of tyrosine kinase receptors including the c-fms One or more of three dimeric PDGF isoforms (PDGF AA, protein receptor for CSF-l and the receptor for fibroblast AB, and BB) are expressed by several cell types within the growth factor, as indicated in Figure 1. Binding of PDGF to extracellular receptor domains initiates a kidney, including resident endothelial, mesangial, vascular smooth muscle and interstitial cells, as well as immigrant progression of events through which PDGF receptors interact mononuclear cells. Recent demonstration that PDGF and with several distinct signaling proteins. Ligand binding appears to PDGF receptor expression are increased at vascular and gb- induce dimerization of individual receptor molecules, and is merular sites in renal allograft rejection and in animal models of tightly coupled to receptor tyrosine seIf-phosphorylation [6]. In renal disease has supported a role for PDGF in the pathogenesis fact, data suggest receptor dimerization is initiated by a or /3 type of destructive proliferation and inflammation [2, 3]. PDGF receptor monomers binding the individual PDGF isoforms The biological activities of individual PDGF isoforms depend for which they have highest affinity (Heldin, this issue). upon their high affinity binding to cell surface receptors expressed on responsive cells. Two molecularly distinct, but highly homolPDGF receptor tyrosine seif-phosphorylation ogous receptors for PDGF molecules, PDGF a and /3 receptors, One of the earliest events initiated by PDGF binding to the were initially distinguished by isoform binding specificities, and receptors' extracellular domain is tyrosine self-phosphorylation subsequently by cDNA cloning, as distinct peptides sharing struc- of two intracellular domain sites (tyrosine residues 751 and 857)

tural and functional characteristics. Individual cell types may express one or both forms of PDGF receptor [4]. Responses to [7]. This self-phosphorylation is mediated by tyrosine kinase distinct PDGF isoforms are mediated through receptor dimers activity intrinsic to the receptor. Data from other tyrosine which may apparently contain one or both receptor types (Heldin, kinase receptors (c-fms, EGF receptor) suggest that self-phosthis issue). The functional studies of PDGF receptor coupling phorylation of one receptor monomer is catalyzed by the

activity of the other monomer's kinase within the dimer pair [81. reviewed here refer largely to PDGF /3 type receptor responses to PDGF BB in murine fibroblasts. It is likely that discrete mecha- If PDGF receptor functions similarly, the importance of ligand-

nisms coupling PDGF /3 receptors to intracellular effector en- induced dimerization in receptor self-phosphorylation is evizymes reflect processes shared among other cell types, including dent, At present, self-phosphorylated PDGF receptors appear mesangial and fibroblast-like cells of the kidney. The molecular to represent the active receptor form which has optimal tybasis for distinct responses to individual PDGF isoforms in cell rosine kinase activity on exogenous substrates and interacts with the downstream effector enzymes indicated in Figure 2. types expressing both a and /3 receptors remain to be resolved. Several observations suggest PDGF receptor tyrosine selfphosphorylation may function as a switch, conferring potential PDGF receptors: Structure and function for interaction with other proteins which participate in PDGFStructural features of PDGF a and /3 receptors contribute to coupled responses. Tyrosine phosphorylated PDGF receptors our current understanding of how these molecules transduce extracellular ligand binding events into intracellular biochemi- have accessible for antibody recognition a cytoplasmic domain epitope which is cryptic in unactivated, non-phosphorylated PDGF receptors [9]. Moreover, self-phosphorylated PDGF receptors stably associate in coprecipitable complexes with © 1992 by the International Society of Nephrology effector enzymes such as phospholipase C (PLC), as discussed

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Daniel and Kumjian: PDGF receptors and PLC activation

PDGF A a

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Fig. 1. PDGF receptor a and f3 types are members of a subclass of tyrosine kinase receptors having an intracellular tyrosine kinase domain interrupted by a peptide 'insert" which is distinct among these proteins. Abbreviations are: PDGF receptors (PDGF R); EGF receptor (EGF R); Insulin receptor (Ins R); CSF-1 receptor (CSF-1 R); fibroblast growth factor receptor (FGF R).

Symbols are: () cysteine rich; (.) cysteine; () C-src homologous tyrosine kinase.

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InsP3 DAG

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below. Finally, receptor dephosphorylation disrupts its capacity for stable association with PLC [10]. PDGF receptor coupled effector enzymes share structural determinants

Efforts of several laboratories have defined participants in PDGF receptor signaling. Several PDGF receptor-coupled signal-generating enzymes have been identified by recognition that they undergo tyrosine phosphorylation in response to PDGF stimulation of intact cells [11—14]. These include phospholipase C7 (PLC), the ras GTPase activating protein (GAP), and the 85 kDa subunit of an enzyme containing phosphatidyl-inositol

are: PLC (phospholipase C,,); c-ras GTPase activator protein (GAP); phosphatidylinositol3-kinase (85 kDa subunit) (P13 K); cellular homologue of the v-raf serine kinase (c-raf); diacylglycerol (DAG); inositol(1 ,4,5)trisphosphate (InsP,).

3-kinase (P13K) activity. As one member of a family of PLC isoenzymes with similar activity, PLC7 is implicated in produc-

tion of second messengers, inositol-(l,4,5)-trisphosphate (InsP3) and diacylglycerol (DAG), which signal downstream calcium release and kinase C activation, respectively [15]. GAP

has been implicated as a major participant in proliferative responses signaled through the cellular homologue of the v-ras oncogene [16]. To date, a clear role for the 3-phosphorylated phosphatidylinositol products of the P13K remains to be elucidated, however, analysis of the function of mutated PDGF f3 receptors has suggested that P13K activation may correlate best with PDGF-induced mitogenic responses [12, 17].

Daniel and Kumjian: PDGF receptors and PLC activation SH31 USH2

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Fig. 3. SH2 and SH3 domains homologous to non-kinase regions of c-src: Components of

(85k) PDGF receptor-coupled effector enzymes. Abbreviations are: Phospholipase C (PLC); c-ras GTPase activator protein (GAP);

v-cr

phosphatidylinositol-3-kinase- (85 kDa subunit) (P13K).

Relevant to the previous suggestion that tyrosine phos- of attachment either between cells or with subcellular matrix phoproteins participate in stable protein-protein associations, it between cultured cells provided some indication that tyrosine has been recognized that PDGF-induced tyrosine self-phos- phosphoproteins may participate in stable protein-protein assophorylation of PDGF receptors promotes their formation of ciations required for cytoskeletal integrity. These dense recogcoprecipitable complexes with each of the effector enzymes nizable structures contain several cytoskeletal proteins includwhich undergo tyrosine phosphorylation (Fig. 2). As the pri- ing vinculin and talin, both of which have been shown to be mary amino acid sequences for PLC7, GAP, and P13K (85 kDa tyrosine phosphoproteins. subunit) have been defined by cDNA sequence, additional In summary, the function apparently implicit to SH2 domains support for some functional importance for their stable associ- represented in these diverse proteins is the capacity to form ation with tyrosine phosphorylated PDGF receptors has been stable interactions with tyrosine phosphoproteins, including seif-phosphorylated tyrosine kinases such as PDGF receptor. generated. PLC7, GAP and P13K share domains which are homologous SH3 domains appear to be shared among proteins which are to regions of the nonreceptor tyrosine kinases of the c-src either recruited to tight association with the plasma membrane, family termed SH2 and SH3 domains [18]. First appreciated to or to proteins such as fodrin which are crucial elements of the determine host range susceptibility for v-src transformation and cytoskeleton. to be the site of mutations in temperature-sensitive transforming mutant versions of v-src, additional interest in these nonPDGF receptor-phospholipase C coupling: Role of proteinkinase domains of the src kinase family was generated with protein association cloning of the transforming oncogene of the CT1O retrovirus, v-crk. This molecule includes SH2 and SH3 domains, and appears to activate native cellular tyrosine kinases in effecting its transforming function [19]. The v-crk protein associates stably with tyrosine phosphoproteins, as do the bacteriallyexpressed SH2 domains of PLC7 and GAP. Proteins having SH3 domains appear to share capacity for interaction with cytoskeletal elements, and elements of the SH3 domain have been implicated in the association of c-src with the plasma membrane. Given the focal concentration of cellular tyrosine phosphoproteins at focal contact sites, it may not be surprising that SH2 and SH3 domains have been identified in several proteins which have the capacity for membrane association and additional proteins which participate in cytoskeletal interactions. Included among these SH3 domain containing proteins are fodrin, myosin ib, a yeast actin binding protein, neutrophil NADPH-oxidase-associated proteins, and c-crk (the cellular homologue of the transforming gene product). Most recently, an actin binding protein, tensin, has been shown to have an intrinsic SH2 domain and to be a substrate for tyrosine kinase activity [20]. The early localization of tyrosine phosphoproteins to focal contacts which serve to anchor cytoskeletal elements at points

Although several effector enzymes are coupled to PDGF receptor activation, certain experimental advantages are realized in addressing the coupling of phospholipase C to PDGF receptor function. PDGF stimulated, PLC-mediated production of InsP3 is easily assayable in intact cells, as it is released from metabolically prelabéled phosphatidylinositol stores. In addition, in vitro assays for inositol phospholipid specific phospholipase C activity may be used to assay PLC activity recovered by phosphotyrosine antibodies or through coprecipitation with PDGF receptors. We have used these advantages to explore the role played by formation of a complex between PDGF receptors and PLC7 in mediating productive PLC7 activation. Despite

this, current understanding emphasizes that several of the

parallel effector systems indicated in Figure 3 participate in the mitogenic responsiveness. Only two methods are currently available for marking cellular PLC molecules as having interacted with PDGF receptors. The PLC protein and activity may be assayed following recovery by phosphotyrosine antibodies from PDGF stimulated cells. Sim-

ilarly, coprecipitation of PLC activity and protein with antibody-recovered PDGF receptors allows identification of the subpopulation of PLC complexed with PDGF receptors under

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Daniel and Kumjian: PDGF receptors and PLC activation

Blocked at 4° C

Demonstrated: 1. PDGF binding 2. A dimerization 3. Kinase activation 4. SeIf-phosphorylation 5. PLC9 association

Proposed Requirements for PLC-mediated InsP Production: 1. Temperature sensitive step (requiring PDGF R occupancy at 370 C) 2. PLCg (-y-P) membrane essociation (free of PDGF A complex)

6. PLCg -phosphorylation 7. Dissociation from PDGF R

Membrane Association

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* Catalytically activated Tyrosine phosphate

Fig. 4. Functional consequences of PDGF association/dissociationireassociation at 4°C on PDGF receptor tyrosine phosphorylation, integrity of receptor-PLC7 coprecipirable complex, and subsequent PDGF-coupled inositol phosphate production.

basal and ligand-activated conditions. In order to define the itinerary of PLC7 molecules participating in PDGF-coupled inositol phosphate production, we have used these tools to follow changes in PLC7 intracellular localization, phosphorylation state, and occupancy in complexes with PDGF receptors.

Only a small fraction of the total pool of PLC7 molecules present in BALB c/3T3 cells actually can be marked as having interacted with PDGF receptors following PDGF stimulation. Under conditions where fully 35% of PDGF receptors undergo activation (as marked by tyrosine phosphorylation), only 5 to 7% of the total cellular pool of PLC7 is either tyrosine phosphorylated or coprecipitable with PDGF receptors 1101. Obviously, both the phosphorylation state of PLC7 molecules and

their presence in complexes with PDGF receptors may be dynamic; steady state accumulation of tyrosine phosphorylated or receptor association PLC7 molecules, may underrepresent the fraction actually participating (see below). Nevertheless, these approaches have allowed us to explore: 1) the functional importance of the coprecipitable PDGF R-PLC

tated tyrosine phosphorylation of PLC and formation of the coprecipitable complex with PDGF R occur at 4°C, albeit at delayed times compared with events occuring at 37°C. Because cellular endocytic processes are interrupted at reduced temperature, the PDGF-bound surface receptors are not internalized.

Remaining on the cell surface, the PDGF bound to these activated receptors may be effectively stripped off receptors, after activated PDGF receptors have phosphorylated PLC7 tyrosine residues. Removal of PDGF bound at 4°C permitted us to determine if tyrosine phosphorylation of PLC7 was sufficient

to commit subsequent inositol phosphate production upon rewarming. Depicted in Figure 4, the PDGF-initiated generation of InsP from cells exposed to PDGF at 4°C proceeds effectively upon rewarming to 37°C, as long as PDGF bound to surface receptors

was retained. When bound PDGF was stripped from surface receptor sites before rewarming, PDGF receptor mediated InsP production was disrupted. Characterizing the effects of acid removal of bound PDGF, we found that the tyrosine phosphate complex in mediating PDGF-coupled InsP production; 2) was initially retained in the majority of PLC7 molecules which whether the PLC7 coprecipitating with PDGF R is tyrosine were phosphorylated by ligand-activated PDGF receptors at phosphorylated; and 3) the subcellular distribution of PLC to 4°C. However, the acid dissociation of bound PDGF completely particulate or soluble fractions following tyrosine phosphoryla- disrupted the coprecipitable PDGF receptor-PLC7 complex tion. The findings suggest the integrity of the PDGF receptor- [101. PLC7 complex is important in activation of PLC7. Also depicted in Figure 4, disruption of the PDGF receptorEarly experiments demonstrated that PIP2-specific PLC ac- PLC7 complexes correlated with the abrupt tyrosine dephostivity coprecipitated with PDGF receptors from PDGF-stimu- phorylation of PDGF receptors upon ligand dissociation. When lated, but not unstimulated, cells [211. Coprecipitation of PLC the dephosphorylation was blocked by pretreatment of the cells activity with ligand-activated PDGF receptors correlated well with the protein tyrosine phosphatase inhibitor, orthovanadate, over varying PDGF doses with PLC-mediated generation of both InsP production upon rewarming and the integrity of the inositol phosphates. Moreover, the PLC activity coprecipitat- PDGF R-PLC7 complex were preserved. These findings may be ing with PDGF receptors accounted for approximately 60% of interpret to show that integrity of the complex containing PLC7 that recovered by phosphotyrosine antibodies from PDGF and PDGF receptors is functionally required at the time of stimulated cells. Thus a significant fraction of the PLC activity rewarming in order for InsP production to follow. This suggests apparently participating in the PDGF-initiated response was that the tyro sine phosphorylation of PLC7 which occurs at 4°C recoverable in a complex with activated PDGF receptors. is insufficient to promote productive PLC7 activation. Further Several features of the interaction between PDGF Rand PLC experiments showed that the receptor-associated PLC7 does have provided useful experimental tools. Both PDGF-medi- not contain phosphotyrosine. This is consistent with the idea

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5. Model of intermediate steps in PDGF receptor activation of PLC. Interruption of the PDGF R-PLC,, complex prior to temperature

transition to 37°C abrogates a step necessary for PLC activation, despite completion of PLC tyrosine phosphorylation before the transition.

residues, at positions 771, 783 and 1254 are substrate sites cides with dissociation of PLC,, from the receptor. Indeed, phosphorylated in vivo and in vitro. When individual tyrosine tyrosine phosphorylation of PLC,, may be required for its residues were substituted with phenylalanine and the mutated that receptor-mediated tyrosine phosphorylation of PLC,, coin-

dissociation from the complex with PDGF receptors. PLC,, molecules expressed, the Y->F783 substitution functionThese findings have led us to conclude that integrity of the ally disabled PLC,, from PDGF receptor activation [221. This receptor-PLC,, complex may be required for a completion of a occurred despite intact in vitro phospholipase activity and

temperature-sensitive process separate from tyrosine phos- demonstration that other tyrosine residues undergo tyrosine phorylation of PLC,,. When PDGF receptor-mediated tyrosine phosphorylation in vivo in response to PDGF receptor activaphosphorylation of PLC,, occurs at 4°C, it appears that some tion. Positioned adjacent to both SH2 and SH3 domains, component function required for inositol phosphate production phosphorylation of Y783 may be important for targeting PLC,, to is not completed, as the model in Figure 5 delineates. Thus, it its membrane active compartment. appears that the stable association of PLC,, with PDGF recepFuture directions tors serves some additional purpose beyond tyrosine phosphorIn summary, it now appears clear that common structural ylation of PLC,, by receptor kinase. Some possible functions for this association include other temperature sensitive processes motifs have been used for overlapping functions in coupling of

such as mobility in the membrane, serine phosphorylation, passing PLC,, to some membrane association determinant or internalization itself.

Data supporting a role for membrane association determinants have been generated by studies addressing the distribution of tyrosine phosphorylated PLC,, to either particulate or soluble compartments. Since the substrate for PLC, phosphatidylinositol phosphates, is a component of the plasma membrane, it may be expected that functionally active PLC,, would become membrane associated, potentially through the SH3 determinant common to other proteins which function in association with the plasma membrane. In fact, the tyrosine phosphorylated PLC,, subpopulation is very inefficiently segregated to a membrane-associated pool at 4°C; two-thirds of the tyrosine phosphorylated PLC,, remain in the soluble fraction, presumably inaccessible to substrate [10]. Structural analysis of the sites of PLC,, tyrosine phosphorylation has provided some additional insight. Three tyrosine

tyrosine kinase receptors to effector enzymes which mediate their responses. Further definition of the distinctive properties of these structural domains, such as the SH2 and SH3 domains, promises to elucidate functional requirements for biochemical and biophysical interactions with tyrosine kinases and additional regulators of their signaling function. At present, functions of proteins which share SH2 and SH3 domains have provided us with good clues that tyrosine phosphoproteins share capacity to stably associate with the SH2 domain, apparently in processes that are controlled through this stable interaction, such as the PDGF receptor coupling to PLC activation. As the G-protein coupled signaling systems have taught us, ultimate definition of the mechanisms will require reconstitution and well defined systems in vitro. Among the most interesting questions posed by the structural motifs common to different signal effector enzymes coupled to

PDGF receptor is that of specificity; in other words, what structural determinants define the capacity of a given SH2

580

Daniel and Kumjian: PDGF receptors and PLC activation

domain-containing protein to participate in PDGF receptor coupled responses. Further definition of structural motifs which

9. KEATING MT, EscoBEDo JA, WILLIAMS LT: Ligand activation

causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation. J Biol Chem 263:12805—

collaborate with phosphotyrosine in defining protein-protein 12808, 1988 interaction is necessary. It is clear, however, that the structural 10. KUMJIAN DA, BARNSTEIN A, RHEE SG, DANIEL TO: Phospholipae information provided by cDNA cloning is providing a rich C gamma complexes with ligand-activated platelet-derived growth factor receptors. J Biol Chem 266:3973—3980, 1991 opportunity for understanding these complex signaling systems. It is hoped that future work will discriminate the distinctive 11. WAHL MI, OLASHAW NE, NISHIBE 5, RHEE SG, PLEDGER WJ, CARPENTER G: Platelet-derived growth factor induces rapid and signaling systems used by differentiated cell types for special-

ized responses to ligands such as PDGF in the kidney and elsewhere.

sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells. Mo! Cell Biol 9:2934—2943, 1989

12. CANTLEY LC, AUGER KR, CARPENTER C, DUCKWORTH B, Gw&-

Reprint requests to Tom Daniel, M.D., MCN S3223, Vanderbilt University, Nashville, Tennessee 37064, USA.

ZIANI A, KAPELLER R, SOLTOFF S: Oncogenes and signal transduction. Cell 64:281—302, 1991 13. COUGHLIN SR. ESCOBEDO JA, WILLIAMS LT: Role of phosphati-

dylinositol kinase in PDGF receptor signal transduction. Science

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