FC Changes in Caveolae Effects of Signal Transduction

The metabolic effects of chronic changes in the FC content of the caveolae can be explained in terms of competition between binding of protein and binding of FC to overlapping sequences within a central (resides 82-101) domain of caveolin, However the content of FC in caveolae also responds both spatially and temporally to physiological changes at the cell surface, in particular to the binding of protein growth factors. Could these changing FC levels be involved in regulating the magnitude and duration of signal transduction and possibly, in the case of branching pathways, its selectivity?

The mechanism of signal transduction via protein growth factors and transmembrane receptor kinases is well understood in outline. Ligand binding is coupled to dimerization of the receptor protein, receptor autophosphorylation, and induction of its activity with downstream kinases, particularly proteins of the c-Src family (see Chapter 6). This leads to dissociation of signaling proteins from the cell surface and eventually, to transcriptional activation of a specific pattern of genes important in mitosis. A consistent feature of ACTH, PDGF, -EGF, VEGF, insulin and insulin-like growth factor-mediated signaling is that receptor autophosphor-ylation is followed by the generation of p(Y14)caveolin, the synthesis of which is

Assembly of cav - FC - PDGFR complex

Ligand binding

^ Activated PDGFR dimer

Relipidated caveolin

Relipidated caveolin

Recruitment of c-Src

CAVEOLIN LIPIDATION CYCLE

Dephosphorylation of caveolin

Phosphorylation of caveolin

LMW-F activity

Fig. 5.5 A multistage caveolin lipidation cycle to explain the roles of FC at different points in signal transduction from caveolae. LMW-PTP = low molecular-weight phosphotyrosine phosphatase (from [121]).

Dissociation of signaling complex

PDGFR

PDGFR

c-Src c-Src

Fig. 5.5 A multistage caveolin lipidation cycle to explain the roles of FC at different points in signal transduction from caveolae. LMW-PTP = low molecular-weight phosphotyrosine phosphatase (from [121]).

mediated by c-Src or a Src-family kinase such as Fyn [50,104-108]. This observation provides prima facie evidence for the existence of signaling complexes containing bound caveolin.

In the case of PDGFR, signal transduction was recently shown to be associated with a loss of FC binding to caveolin, assayed either by co-purification of 3H-FC with caveolin, or by the formation of stable crosslinks between caveolin and a photoactivable FC analogue, FCBP [50]. A comparable change occurs when VEGF activates its receptor in human aortic endothelial cells, and confirms that the association of FC with caveolin is quite labile. Even though <1 % of caveolin was associated with PDGFR, a maximum of 70-80% of caveolin-associated FC was lost in the course of signal transduction. This is consistent with the concept that FC bound to caveolin has higher-order effects on membrane FC in caveolae. Reassembly of the PDGFR signaling complex requires the re-establishment of FC levels in the caveola (Fig. 5.5). Studies of the time course of signaling in the presence and absence of inhibitors of specific kinases and phosphatases, and substitution within caveolae of mutant caveolins with altered polarity and FC binding properties, were carried out.

The yield of caveolin-associated 3H-FC or -FCBP under different conditions was assayed. Recovery of the FC content of caveolae following exposure to PDGF took 30-40 min at 37 °C. The cell became responsive again to PDGF only after the FC

content of its caveolae was completely recovered. If the activity of the caveola-associated low molecular-weight phosphotyrosine phosphatase was inhibited by hydrogen peroxide [121], p(Yi4)caveolin accumulated and the caveola did not recover its reactivity with PDGF. If caveolin(S80A), which binds FC more tightly than the native protein [50] was overexpressed, then although PDGF bound normally to its receptor, FC was not displaced from this caveolin, and the signal was not propagated from the PDGF receptor (PDGFR) to c-Src and to caveolin. These data suggest that, at different steps, FC has positive and negative roles. The presence of FC is required for the assembly of the transmembrane signaling complex; after ligand binding, loss of FC is required for signal transduction. Following signal transduction, FC re-enters the caveola. These observations are incorporated into Fig. 5.5:

1. A signaling complex containing at a minimum caveolin, FC, a transmembrane receptor kinase, a Src-family non-receptor kinase is first assembled within caveolae. At least in SMC, caveolin is constitutively phosphorylated at S80, influencing FC binding to the cav(82-101)

2. Addition of ligand leading to receptor and downstream receptor kinase activation displaces FC from its binding site. Mutant Caveolin(S80A), which cannot be phosphorylated, does not release FC and signal transduction is inhibited.

3. Loss of FC from caveolin precedes p(Y14)-caveolin synthesis mediated by c-Src or Src-family kinase. Since the level of p-caveolin, which is normally low, is dynamically regulated by caveolar LMW-PTP, the rise in p-caveolin which follows the loss of FC must be the result of either the dissociation of this phosphatase, or its inhibition in situ. These alternatives have not yet been distinguished. p-Caveolin synthesis in response to PDGF was not associated with any detectable disassembly of caveolin oligomers, or withdrawal of caveolin from the cell surface. This is in contrast to what occurs when p(Y14)-caveolin is chronically increased - for example by vanadate, a nonspecific phosphotyrosine phospha-tase inhibitor.

4. After dissociation of both FC and downstream kinases, LMW-PTP is reactivated, and reduces p-caveolin levels to baseline. Levels of total caveolin continue unchanged.

5. FC levels in caveolae (and those of FCBP in cells equilibrated with this photo-activable FC analogue) are restored only after p-caveolin is hydrolyzed. In cells exposed to peroxide, phosphocaveolin levels were maintained at their maximum; FC was not restored to caveolae, and the cells remained insensitive to PDGF.

6. Finally (at ca. 30 min following initial exposure to PDGF) the cell regains responsiveness to ligand.

These data indicate that the overall equilibrium between FC-associated and FC-free caveolin in caveolae is influenced by phosphorylation of both receptor protein and caveolin at different points in the signaling reaction.

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