Structure-function of CD36 and importance of fatty acid signal transduction in fat metabolism

Abstract

CD36 (cluster of differentiation 36) is a scavenger receptor that functions in high-affinity tissue uptake of long-chain fatty acids (FAs) and contributes under excessive fat supply to lipid accumulation and metabolic dysfunction. This review describes recent evidence regarding the CD36 FA binding site and a potential mechanism for FA transfer. It also presents the view that CD36 and FA signaling coordinate fat utilization, a view that is based on newly identified CD36 actions that involve oral fat perception, intestinal fat absorption, secretion of the peptides cholecystokinin and secretin, regulation of hepatic lipoprotein output, activation of beta oxidation by muscle, and regulation of the production of the FA-derived bioactive eicosanoids. Thus abnormalities of fat metabolism and the associated pathology might involve dysfunction of CD36-mediated signal transduction in addition to the changes in FA uptake.

Keywords: CCK; FA binding; VLDL; calcium; chylomicron; eicosanoid; fat taste; phospholipase; secretin.

Figure 1. Schematic representation of CD36 binding domains and post-translational modifications

CD36 has two short intracellular domains at both termini, two transmembrane segments and a large extracellular domain with a hydrophobic sequence where lipid ligands bind. The C-terminus can associate with Src tyrosine kinases, which initiate most of CD36 mediated signal transduction. The diagram highlights the post-translational modifications of the protein including its glycosylation, palmitoylation, ubiquitination, phosphorylation and acetylation. These modifications influence CD36 trafficking and function in FA uptake and signal transduction (see text for details). Both N- and C- termini contain two palmitoylation sites which localize CD36 to membrane lipid rafts. Binding sequences for CD36 ligands are aligned to the backbone of CD36 and the FA-binding site K164 is highlighted with a dotted line [for details see text and (44)].

Figure 2. The FA binding site on CD36 and its access to a transport tunnel within the molecule

The structure of human CD36 exofacial domain (K36 – G436) was modeled using the Phyre2 prediction server (41) based on the recently reported crystal structure of the CD36 family member LIMP2 (68). 98% of residues modeled at >90% confidence. Fig 2-A: Ribbon model of CD36 structure highlighting the SSO-target residue K164. Also highlighted is K334 a secondary and rare SSO target (44). Figure 2-B: Detail of the CD36 structure showing the FA (oleic acid) docking site using the SwissDock server (30) (Full fitness -2409.97 kcal/mol; estimated ΔG -8.59kcal/mol). Figure 2-B: Surface hydrophobicity is indicated, with blue denoting hydrophilic residues and red hydrophobic residues. The FA is shown to dock within a hydrophobic pocket at the top of CD36 near helix 5 with the FA carboxylic tail in close proximity to lysine 164 (green residue), previously shown to bind the oleate derivative SSO (44). The FA pocket is shaped into a sliding groove that heads towards the tunnel present within the protein structure, modeled based on the one identified by the crystal structure of LIMP2 (68). The glutamic acid residue 335 (magenta color) is positioned at the site of FA entry into the tunnel. K164 might be important for correct positioning of the FA within the pocket possibly allowing it to slide into the tunnel, and for inducing a conformational change in the protein that initiates signaling. Fig. 2-C: A vertical slice through the CD36 modeled structure showing the rear side of the tunnel which is formed mainly by beta strand 21 and outlets close to the plasma membrane. The arrows show predicted direction of FA transport. Our working hypothesis is that the FA interacts with K164 within the hydrophobic groove which positions it to slide with the acyl chain leading the way into the tunnel. The outlet of the tunnel is likely buried in the charged layer of the membrane which would facilitate transport of the hydrophobic acyl chain through the bilayer.

Figure 3. Oral perception of fat

The accumulating evidence supports gustatory cues in fat perception. Dietary fat is composed mainly of triglycerides (TG) but the nutrient sensed on the tongue is the FA released from TG digestion by lingual lipase. The FA interacts with CD36 on taste bud cells in the circumvalate and fungiform papillae (back and sides of the tongue, respectively) to induce intracellular calcium release from the ER. This in turn triggers calcium flux from membrane store operated calcium (SOC) channels leading to neurotransmitter release. The two FA receptors identified on taste bud cells in rodents and humans are CD36 and GPR120 (Table 1) and both were shown to impact sensitivity to oral perception of dietary fat. CD36 is the physiologically functional FA receptor with GPR120 acting to amplify signaling at high FA levels [see text and (69)]. TG: triglyceride, FA: fatty acid, PLC: phospholipase C, PIP2: phosphatidyl inositol-biphosphate, IP3: Inositol triphosphate or inositol 1,4,5-triphosphate, DG: diacylglycerol.

Figure 4. Working model of the molecular steps that may be involved in how intestinal CD36 facilitates FA uptake and chylomicron formation

Panel A: CD36 expressed on the apical side of enterocytes of the proximal intestine interacts with fatty acids (FA) released from the digestion of dietary triglycerides. During the early phase of digestion when the FA concentrations are relatively low CD36 facilitates FA uptake probably by internalizing the FA within vesicles derived from membrane lipid rafts. The CD36-mediated FA uptake associates with intracellular signaling to promote events that facilitate chylomicron assembly. CD36 signaling is initiated in most cases via the Src kinases that associate with the C terminus of CD36 and downstream would involve the extracellular regulated kinase (ERK1/2). CD36 signaling may be important for phosphorylating proteins required to coordinate ER processing of prechylomicron vesicles (PCTV). CD36 has also been identified in the protein complex required for formation of the PCTV (82). Recent data indicate that CD36 signaling may be mediated by inositol triphosphate (IP3)-induced release of ER calcium (see Fig. 3). ER calcium release promotes membrane CD36 localization and also induces calcium influx via store operated calcium channels. The sustained increase in intracellular calcium could influence multiple events related to lipid processing or secretion. Panel B) CD36 is downregulated by FA via its ubiquitination on its carboxyl terminus which targets it to lysosomal degradation. This feedback loop may work to reduce CD36 function in the presence of excess FA supply. Inside the enterocytes the FA-induced decrease in CD36 associates with reduced activation of ERK1/2 which may serve to upregulate abundance of the microsomal triglyceride transfer protein (MTTP). Adapted from (2).