STRA6, a cytokine signalling receptor activated by holo-retinol-binding protein

We recently discovered that STRA6 functions as a surface signalling receptor⁽ Reference Berry, Jacobs and Marwarha ^{13 ,} Reference Berry, Jin and Majumdar ^{16 –} Reference Marwarha, Berry and Croniger ^{19 )}. Such receptors are activated by extracellular cytokines, hormones and growth factors and they transduce signalling cascades by activating protein kinases termed Janus kinases (JAK) and their associated transcription factors called signal transducers and activators of transcription (STAT). Upon their activation, STAT move to the nucleus where they regulate the transcription of specific target genes which contain STAT response elements in the regulatory regions⁽ Reference Aaronson and Horvath ^{20 –} Reference Darnell, Kerr and Stark ^{22 )}.

We found that treatment of STRA6-expressing cells with holo-RBP4 leads to phosphorylation of STRA6, in turn triggering recruitment and activation of JAK2 and, in a cell-specific manner, STAT3 or STAT5⁽ Reference Berry, Jin and Majumdar ^{16 )}. STRA6 recruits STAT through an amino acid sequence in the receptor's intracellular domain that contains a consensus phosphotyrosine motif⁽ Reference Pasutto, Sticht and Hammersen ^{12 )}. Holo-RBP4 thus functions as a classical cytokine to activate a STRA6/JAK2/STAT3/5 pathway. Remarkably, unlike other cytokine receptors, STRA6 is activated not simply by binding its ligand but by the act of transporting retinol. Consequently, STRA6 signalling critically depends on STRA6-mediated retinol transport. Our data further showed that, in turn, retinol transport cannot proceed if STRA6 phosphorylation is impaired⁽ Reference Berry, Jacobs and Marwarha ^{13 ,} Reference Berry, O'Byrne and Vreeland ^{18 )}. The two functions of STRA6 are thus critically inter-dependent. Moreover, we found that STRA6 not only binds holo-RBP4 but also directly associates with the intracellular RBP termed CRBP1. Hence, the receptor does not transport vitamin A into the cytoplasm but, instead, directly transfers it from extracellular RBP4 to intracellular CRBP1, bypassing the need for the lipophilic vitamin to dissociate into the aqueous milieu of the cytosol⁽ Reference Berry, O'Byrne and Vreeland ^{18 )}. Upon binding retinol, CRBP1 dissociates from STRA6 and delivers the vitamin to a retinol-metabolizing enzyme. One such enzyme is Lecithin:retinol acyltransferase, which catalyses the conversion of retinol to its storage species retinylesters. Lecithin:retinol acyltransferase receives its substrate directly from CRBP1⁽ Reference Herr and Ong ^{23 )}, unloads retinol from CRBP1 and, by metabolizing it, maintains an inward-directed retinol concentration gradient allowing continuing retinol uptake⁽ Reference Marwarha, Berry and Croniger ^{19 ,} Reference Amengual, Golczak and Palczewski ^{24 )}. As retinol transport is required for activation of STRA6 signalling, both the cellular RBP and the retinol metabolizing enzyme are necessary for enabling receptor phosphorylation⁽ Reference Berry, O'Byrne and Vreeland ^{18 ,} Reference Marwarha, Berry and Croniger ^{19 )}. A model for the mechanism of action of STRA6 is shown in Fig. 1.

Fig. 1. (Colour online) Model for the retinol-binding protein (RBP4)/STRA6 pathway. STRA6 binds extracellular holo-RBP4 and transports retinol to receptor-associated cellular retinol-binding protein (CRBP). Retinol transfer activates STRA6, thereby triggering a signalling cascade mediated by the kinase Janus kinases 2 (JAK2) and its associated transcription factors signal transducers and activators of transcription 3 (STAT3) or STAT5. Activated STAT translocates to the nucleus where it induces target gene expression. Upon binding retinol, CRBP dissociates from STRA6 and delivers retinol enzymes that can metabolise it.

As mentioned earlier, holo-RBP4 circulates in blood bound to TTR. We found that although TTR does not interfere with the movement of retinol into cells by free diffusion, it prevents holo-RBP4 from binding to STRA6. TTR thus inhibits both STRA6-mediated retinol transport and STRA6-initiated signalling⁽ Reference Berry, Croniger and Ghyselinck ^{17 )}. As the binding affinity of holo-RBP4 to TTR and STRA6 are similar⁽ Reference Kawaguchi, Yu and Honda ^{10 ,} Reference Noy, Slosberg and Scarlata ^{25 )}, STRA6 can function only when its expression level in particular cells is very high, allowing it to locally compete with TTR for holo-RBP, or when serum level of holo-RBP4 exceeds that of TTR. The high expression level of STRA6 in the retinal pigment epithelium thus allows the receptor to significantly contribute to vitamin A uptake by the eye⁽ Reference Ruiz, Mark and Jacobs ^{15 )}. Interestingly, it was reported that serum level of RBP4 is elevated in obese mice and human subjects⁽ Reference Berry, Croniger and Ghyselinck ^{17 ,} Reference Yang, Graham and Mody ^{26 ,} Reference Berry, Soltanian and Noy ^{27 )} but the level of TTR is not⁽ Reference Berry, Croniger and Ghyselinck ^{17 )}. Under these circumstances, the high RBP : TTR ratio in blood may lead to hyperactivation of STRA6. It was also reported that increasing the blood level of RBP4 either by feeding mice a high fat-containing diet or by direct injection of the protein causes insulin resistance⁽ Reference Berry, Croniger and Ghyselinck ^{17 ,} Reference Yang, Graham and Mody ^{26 –} Reference Graham, Yang and Bluher ^{28 )}. These observations raise the intriguing possibilities that RBP-induced activation of STRA6 may be involved in regulation of insulin responses, and that such an activity may underlie the well-established but incompletely understood link between obesity and insulin resistance.

Involvement of retinol-binding protein/STRA6 signalling in regulation of insulin responses

The discovery of the holo-RBP/STRA6 signalling cascade and the observations that this pathway activates STAT suggest a possible mechanism through which RBP4 may control insulin responses. A prominent STAT target in the insulin-responsive tissues adipose tissue and muscle is the gene that encodes suppressor of cytokine signalling 3⁽ Reference Starr, Willson and Viney ^{29 )}, a potent negative regulator of the insulin receptor. Indeed, we found that holo-RBP4 suppresses insulin receptor in cultured cells and that the effect required activation of STRA6, JAK2 and STAT5 as well as up-regulation of suppressor of cytokine signalling 3⁽ Reference Berry, Jin and Majumdar ^{16 )}. In accordance, administration of RBP4 to mice activated STRA6, JAK2 and STAT5, up-regulated suppressor of cytokine signalling 3 and decreased the phosphorylation status of insulin receptor in adipose tissue and muscle, but not in liver, a tissue that does not express STRA6⁽ Reference Berry, Jin and Majumdar ^{16 )}. Additional experiments showed that STRA6-null mice are completely protected from insulin resistance caused by administration of RBP4 and partially protected from obesity-induced glucose intolerance. It was recently reported in regard to this that even partial reduction on STRA6 only in adipose tissues improves insulin responsiveness in obese mice⁽ Reference Zemany, Kraus and Norseen ^{30 )}. These observations conclusively demonstrate that STRA6 mediates RBP4-induced suppression of insulin signalling and that elevated RBP4 levels in blood of mice contribute to the well-documented but incompletely understood link through which obesity leads to insulin resistance.

Concluding remarks and future directions

The discovery that retinol in conjunction with its blood carrier RBP4 can control cellular functions by activating STRA6-mediated cell signalling reveals a new mechanism by which vitamin A exerts its biological functions. The observations show that holo-RBP4/STRA6 signalling regulates insulin responses, but the complete spectrum of the biological activities of the path remain to be clarified. Interestingly, it has been reported that STRA6 is up-regulated in several human cancers⁽ Reference Szeto, Jiang and Tice ^{11 )}. STRA6 and its associated components may thus be involved in oncogenic activities. While available information indicates that STRA6 can trigger a JAK2/STAT3/5 cascade, it is possible that, like other cytokine receptors, STRA6 can also activate other signalling pathways. The complete spectrum of the signalling networks that may be activated by this receptor and cross-talk between this pathway and others remain to be explored. The observations open a new window on vitamin A biology, and they suggest that STRA6 may be a druggable target for novel approaches for therapy of metabolic disease and perhaps other pathologies.