Endosomes, receptor tyrosine kinase ... - Semantic Scholar

Bioscience Reports, Vol. 15, No. 6, 1995

Endosomes, Receptor Tyrosine Kinase Internalization and Signal Transduction John J. M. Bergeron, T M G. M. Di Guglielmo, 2 Patricia C. Baass, 1 Francois Authier, 4 and Barry I. Posner 3 Received September 8, 1995 Upon the binding of insulin or epidermal growth factor to their cognate receptors on the liver parenchymal pl~Sinalemma signal transduction and receptor internalization are near co-incident. Indeed, the rapidity and-extem 9f ligarid mediated receptor internalization into endosomes in liver as well as other organs predicts that signal transduction is regulated at this intracellular locus. Although internalization has been thought as a mechanism to attenuate ligand mediated signal transduction responses, detailed studies of internalized receptors in isolated liver endosomes suggest an alternative scenario whereby selective signal transduction pathways can be accessed at this locus.

KEY WORDS: Endosome; insulin; internalization; receptor; signal transduction; tyrosine kinase.

INTRODUCTION

Extracellular hormones and growth factors regulate cell metabolism, growth, differentiation and mitogenesis by interacting with plasma membrane located receptors. One class of these receptors are type I transmembrane proteins containing extraceltular domains that bind soluble polypeptide hormones or growth factors and cytosolic domains that induce, via an endogeneous tyrosine kinase activity, a cascade of intracellular signaling events. However, upon ligand binding, receptors are rapidly internalized into a series of non-lysosomal compartments collectively called the endosomal apparatus (Bergeron et al., 1985). Detailed studies on a number of receptor tyrosine kinases suggest that internalization, rather than simply serving as a mechanism for attenuation, may also allow for selected and regulated signal transduction from intracellular compartments. 1 Departments of Anatomy and Cell Biology, 2Biochemistry, and 3Medicine, McGill University, Montreal, P.Q., Canada H3A 2B2 and 4INSERM U30, H6pital des Enfants Malades, Paris; France. s To whom correspondence should be addressed. 411 0144-8463/95/1200-0411507.50/09 1995PlenumPublishingCorporation

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Bergeron, Guglielmo,Baass, Authier and Posner INTRACELLULAR RECEPTORS FOR INSULIN

We have attempted to evaluate the relationship between receptor internalization and signal transduction. Our studies commenced with insulin and one of its major target organs for the maintenance of blood glucose homeostasis, liver parenchyma. Our interest arose out of a finding that insulin receptors were found to be present in both plasma membrane and purified Golgi fractions (Bergeron et al., 1973; Bergeron et al., 1978; Posner et al., 1978). Since these initial studies were largely based on preparative subcellular fractionation, we attempted to pursue an alternative strategy that would help us visualize insulin and hopefully its receptor. We had found that by the technique of electron microscope (EM) radioautography we could visualize insulin receptors by the specific binding of 125I-radiolabeled insulin to Golgi fractions in vitro (Bergeron et al., 1978). This established conclusively that such binding was not to plasma membrane contaminants. We next attempted to evaluate if we could use electron microscopy to visualize 125I-insulin specific binding sites in vivo. Here whole animals were injected with tracer doses of 125I-insulin and control animals were injected with the same dose of lzSI-labeled insulin and an excess of unlabeled insulin. After rapid removal of unbound hormone by a saline wash, whole body fixation and cross linking of 125I-radiolabeled insulin was carried out. This led, at the EM level, to a clear and direct visualization of specific binding sites at the liver parenchymal plasma membrane (Bergeron et al., 1977). EM radioautography is a quantitative technique and was pursued to (i) identify target organs for several hormones and growth factors at the morphological level and (ii) evaluate over a timecourse, after hormone administration, the intracellular localization of radiolabel. In the former case, this technique has been instrumental in evaluating established and new targets for lesI-insulin (Bergeron et al., 1980), EGF (Martineau-Doiz6 et al., 1988), calcitonin (Warshawsky et al., 1980), prolactin (Bergeron et al., 1983), and recently TGF/3 (Dickson et al., 1995). Rapid polypeptide hormone internalization into non-lysosomal compartments was directly visualized by this technique (Bergeron et al., 1979). In our first study we showed by combining the techniques of EM radioautography with acid phosphatase cytochemistry that ~zsI-insulin accumulated in Golgi associated vesicles of an identical morphology to vesicular compartments originally identified in isolated Golgi fractions in which we visualized 125I-insulin specific binding. After extending the observations to lZSI-prolactin binding sites (Bergeron et al., 1983) we decided to address whether this rapid intracellular sequestration was related to a modulation of receptor bioactivity. Two approaches were pursued. The first was to obtain a greater clarification of the Golgi associated compartments and the second was to assess quantitative methods for measuring receptor activity.

INTRACELLULAR COMPARTMENTS HARBORING INTERNALIZED POLYPEPTIDE KINASE RECEPTOR COMPLEXES

The existence of non-lysosomal compartments enriched in internalized insulin which sedimented by subceUular fractionation in isolated liver Golgi

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fractions and which were observed in situ close to stacked saccules of the Golgi apparatus was originally considered unprecedented. More extensive subcellular fractionation of hepatic Golgi fractions along with the analytical fractionation of light mitochondrial fractions revealed vesicle heterogeneity in compartments harboring intracellular polypeptide hormone receptors (Khan et al., 1981; 1982). By using methodology originated by Courtoy and colleagues (1984) to modify the density of intracellular organelles of endocytic origin we could completely separate the vesicular components internalizing 125I insulin from Golgi apparatus (and lysosomal) markers (Kay et al., 1984). In order to account for the main features of this compartment which concerned its co-isolation and near identical density with Golgi apparatus (Bergeron et al., 1986) and content of intraluminal particles, we postulated that this endocytic compartment in liver must be highly enriched in lipoprotein particles. We have indeed visualized directly the colocalization of internalized horse radish peroxidase with apolipoprotein E in juxta Golgi vesicles of the same morphology by double gold label EM immunolabeling (Dahan et al., 1994) as those previously analyzed by EM radioautography as internalizing 125I insulin and other polypeptide hormones (Bergeron et al., 1979). We and others-have called this novel non-lysosomal endocytic compartments the endosomal apparatus (Bergeron et al., 1985). A sequential transport of 125I-insulin and 125I-prolactin through endosomal compartments which differed in their density on Percoll gradients but especially of increased size by differential centrifugation was observed and considered "early" and "late" endosomal compartments (Bergeron et al., 1986; Khan et al., 1986). Two noteworthy observations were made about this time. Firstly, it was found that in contrast to prolactin, insulin was rapidly degraded immediately after internalization (Posner et aL, 1982, Bergeron et aL, 1986) by a mechanism which was chloroquine sensitive and therefore dependent on acidification (Posner et al., 1982). Secondly, our studies were extended to the EGF receptor in order to evaluate the relationship between internalization and receptor kinase activity.

E N D O S O M A L SIGNAL T R A N S D U C T I O N

Male rat liver parenchymal cells harbor near equal numbers for insulin and epidermal growth factor receptors (Lai et al., 1989a; Burgess et al., 1992). In conjunction with Cohen and Fava (1985) we uncovered that after internalization, receptor tyrosine kinase (RTK) activity was maintained for the EGF receptor (Kay et al., 1986; Lai et al., 1989a,b). This was also found for the insulin receptor (Khan et al., 1986). Most remarkable however, was the finding that the Vmax/Km of exogenous kinase activity and autophosphorylation activity of the insulin receptor after rapid internalization into endosomes was higher than that of the receptor at the peak time of ligand induced activation on the plasma membrane (i.e. at 30 sec) (Khan et al., 1989; Burgess et al., 1992). Activation of the insulin receptor tyrosine kinase is effected by autophosphorylation on adjacent tyrosine residues in the catalytic domain (Tornqvist et al., 1988).

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Remarkably however is that the insulin receptor kinase, after ligand activation, is considered ligand independent in its kinase activity (Rosen et al., 1983). Hence the elevated Vmax/Km of the kinase activity of the internalized endosomal insulin acceptor clearly predicted signal transduction in the endosome (Khan et aL, 1986; 1989). For the EGF receptor, direct labeling after 32p injection into whole animals revealed that its phosphotyrosine content actually increased after internalization. Furthermore, after immunoprecipitation of the tyrosine phosphorylated EGF receptor from endosomal fractions, a 55 kDa protein was co-precipitated and was termed pyp55 (Wada et al., 1992). This 55 kDa protein was subsequently identified by Donaldson and Cohen (1992) as the major in vivo substrate of the EGFR during mouse development in several EGF RTK enriched target organs. Shortly thereafter this 55 kDa protein was uncovered as the adaptor protein SHC (Ruff-Jamison et al., 1993, Di Guglielmo et al., 1994). This clearly established the endosome as a locus for signal transduction. As shown in Fig. 1, purified plasma membrane and endosomal fractions exhibit high levels of tyrosine phosphorylated proteins especially in endosomes as assessed by immunoblotting with anti-phosphotyrosine antibodies. A complex of phosphotyrosine modified EGF receptor, its major substrate SHC, as well as the adaptor protein GRB2 and associated mSOS (the GDP:GTP exchange factor for ras) has been identified in endosomes (Di Guglielmo et al., 1994). Indeed using phosphopeptide specific antibodies it was even uncovered that the endosomal internalized EGF RTK was phosphorylated at tyrosine 1173 in vivo. Pursuing the compartmentalization of signal transduction from the EGF RTK in liver parenchyma, we also uncovered a cytosolic pool of tyrosine phosphorylated SHC. Here tyrosine phosphorylated SHC was in association with GRB2 and mSOS. In order to assess the relevance to the ras activation of the MAP kinase pathway, we evaluated ras activation of raf-1 by assessing its mobility shift in SDS-PAGE. We found a prolonged timecourse of ras activation and SHC activation. These studies have led to a model (Fig. 2) whereby EGF

Fig. 1. EGF dependent tyrosine phosphorylation of proteins in plasma membrane and endosomal fractions. Plasma membrane and endosomal fractions were isolated from rat liver bomogenates 0-60min after the intraportal injection of EGF. One hundred p~g of plasma membrane fraction protein and 50 jxg of endosomal fraction protein were separated by SDS-PAGE and immunoblotted with antibodies raised to phosphotyrosine. The mobility of standard molecular mass markers are indicated on the left. The EGF receptor (170kDa) and SHC (55kDa) have been identified (Di Guglielmo et al., 1994).

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~RAS

SHC

#

'---.

P.

- o t h e r s i g n a l i n g pathways? - receptor clearance?

Fig. 2. Model for EGFR signal transduction in rat liver. The ligand induced activation of the EGF receptor leads to the recruitment of SHC, its tyrosine phosphorylation and the recruitment of the adaptor protein GRB2 (in association with mSOS) to the plasma membrane where they may interact with ras. Coincident with recruitment of these signaling constituents is ligand induced receptor internalization. With its tyrosine kinase tail cytosolically exposed, the endosomal EGF receptor continues to phosphorylate SHC and recruit GRB2/mSOS. It is postulated that both the plasma membrane and the endosomally located EGF receptor generate the pool of complexed SHC, GRB2 and mSOS that is found in the cytosol. The endosomal EGF receptor may interact with yet unidentified signaling molecules at this locus to effect other cellular responses (figure taken from Di Guglielmo et al., 1994).

e n d o s o m a l p h o s p h o t y r o s i n e m o d i f i e d S H C is a s o u r c e of the cytosolic p o o l of a c t i v a t e d S H C and a c t i v a t i o n o f ras. This p r o v i d e s a r a t i o n a l e for S H C as an a d a p t o r m o l e c u l e m e c h a n i s t i c a l l y distinct t o t h a t of G R B 2 / m S O S in d i r e c t a s s o c i a t i o n with a c t i v a t e d E G F R T K at t h e p l a s m a m e m b r a n e . P a r a d o x i c a l l y , for t h e case of t h e insulin r e c e p t o r , a l o w e r p h o s p h o t y r o s i n e c o n t e n t was o b s e r v e d a f t e r i n t e r n a l i z a t i o n i n t o e n d o s o m e s (Burgess et al., 1992). C o n s i d e r i n g its e l e v a t e d level of k i n a s e a c t i v i t y h o w e v e r , a d e p h o s p h o r y l a t i o n event i m p l i e d that a c t i v a t i o n m a y b e similar to t h a t o f t h e e s t a b l i s h e d m e c h a n i s m o f a c t i v a t i o n o f c-src ( B u r g e s s et al., 1992). I n d e e d c-src is k n o w n to b e e n d o s o m a l in fibroblasts ( K a p l a n et al., 1992) a n d it is at this locus t h a t t h e a c t i v a t i n g

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dephosphorylation reaction (at phosphotyrosine 527) likely takes place (Kaplan et aL, 1995).

Consequently we searched for such tyrosine phosphatase (PTPase) activities and uncovered potent PTPase activity on the cytosolic surface of endosomes (Faure et al., 1992). More recently, we have observed that the stable peroxovanadium derivative bpvPhen is targeted in vivo to an endosomal PTPase with apparent specificity for the Insulin RTK. Indeed, in animals where bpvPhen but no insulin was administered, conditions were selected whereby only the endosomal insulin RTK would be activated. This led to activation of an insulin specific signal transduction pathway via IRS-1 the major substrate of the activated insulin RTK (Bevan et al., 1995). In fat cells it is clear that the endosomal insulin RTK is the preferential site of phosphorylation of IRS-1 in association with PI3' kinase (which are themselves in a distinct intracellular compartment) (Kublaoui et aL, 1995). Indeed recent studies have pointed to endosomal signal transduction for the internalized activated PDGF receptor RTK (Kapeller et al., 1993), as well as the NGF receptor RTK (Ehlers et al., 1995). A major rationale for the internalization of RTKs into endosomes is to regulate the extent and perhaps selectivity of signal transduction. An important function of the endosome is to regulate sorting of internalized receptors: Insulin receptors recycle back to the plasma membrane more readily than E G F receptors (Lai et aL, 1989b). An event for the insulin receptor which may be relevant to both insulin RTK activity and receptor sorting is that of endosomal insulin degradation. Following from our initial findings that insulin was selectively degraded (as compared to other ligands) after entry into the endosomal apparatus we first evaluated with isolated endosomes whether such a selective endosomal proteinase existed (Doherty et aL, 1990). We then set out to purify the proteinase and uncovered that it selectively bound and degraded insulin at low pH and that it was unrelated to proteases previously described (Authier et al., 1994; 1995).

CONCLUSION In summary, upon hormone and growth factor binding, receptor tyrosine kinases are rapidly internalized and concentrated in endosomes. Here selective signal transduction takes place prior or perhaps coincident with sorting of the receptors either back to the plasma membrane or to lysosomes (via multivesicular endosomes) for degradation. The elucidation of the molecules in endosomes which regulate signal transduction and receptor sorting at this locus is eagerly awaited.

ACKNOWLEDGEMENTS

JJMB was supported by a studentship from the F C A R of Quebec. GMDG was supported by a Steve Fonyo studentship of the NC1 of Canada. Supported by operating grants from the NCI and MRC of Canada to BIP and JJMB.

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