Identification of the growth hormone-releasing peptide binding site in CD36: a photoaffinity cross-linking study

Abstract

The GHRPs (growth hormone-releasing peptides) are a class of small synthetic peptides known to stimulate GH release through binding of a G-protein-coupled receptor (designated GHS-R). We have found that hexarelin, a hexapeptide member of the GHRPs, binds to another protein identified as CD36, a scavenger receptor that is expressed in various tissues, including monocytes/macrophages and the endothelial microvasculature. CD36 is involved in the endocytosis of oxLDL (oxidized low-density lipoprotein) by macrophages, and in the modulation of angiogenesis elicited by thrombospondin-1 through binding to endothelial cells. To define the binding domain for hexarelin on CD36, covalent photolabelling of CD36 followed by enzymic and chemical degradation of the photoligand-receptor complex was performed. A 8 kDa photolabelled fragment corresponding to the CD36-(Asn132-Glu177) sequence has been identified as the hexarelin-binding site. Chemical cleavage of this fragment with CNBr resulted in the release of the free ligand, suggesting that Met169 is the contact point for the ligand within the receptor binding pocket. We conclude that the binding domain for hexarelin on CD36 overlaps with that for oxLDL, which corresponds to residues Gln155-Lys183 of CD36. Hence hexarelin might interfere with the CD36-mediated uptake of modified lipoproteins by macrophages. This may contribute, at least in part, to the anti-atherosclerotic effect of GHRPs in apolipoprotein E-deficient mice.

Figure 1. Western blot analysis of CD36 expression

Expression of the CD36 protein was assessed in membrane preparations from human and mouse hearts, mouse monocytes/macrophages, human platelets and HEK293/hCD36 cells. Total protein (60 μg) was separated by SDS/7.5%-PAGE and probed with specific anti-CD36 antibodies. Immunoblots for cardiac membranes, macrophages, platelets and HEK293/hCD36 cells are shown above the bars. Relative protein levels for CD36 are expressed as relative abundance (%) compared with that in the heart membrane preparations (set at 100%).

Figure 2. Photoaffinity cross-linking of [¹²⁵I]Tyr-Bpa-Ala-hexarelin with CD36

(A) Amino acid sequences of hexarelin and of its photoactivatable derivative used in photoaffinity labelling studies. (B) Primary amino acid sequence of the rat CD36 receptor. Bold circles indicate recognition residues for Endo Glu-C. Black filled circles indicate methionine residues, which are sensitive to CNBr treatment. The oxLDL-binding site is represented by grey filled circles. Putative glycosylation sites are indicated with Y-shaped pointers. Autoradiography (inset) of the labelled receptor was achieved by incubation of rat cardiac membranes as a source of CD36 (4 mg/ml) with 0.33 nmol/l [¹²⁵I]Tyr-Bpa-Ala-hexarelin in Tris/HCl buffer at 22 °C for 1 h. The tubes were exposed to UV light (365 nm), treated or not with Endo F (50 m-units/μg of protein) and submitted to electrophoresis on a 7.5% (w/v) acrylamide gel. Arrows indicated the native radiolabelled receptor (88 kDa) and its deglycosylated form (55 kDa).

Figure 3. Competition curves for endogenous CD36 ligands and GHRPs for binding of [¹²⁵I]Tyr-Bpa-Ala-hexarelin to CD36

A fixed concentration of [¹²⁵I]Tyr-Bpa-Ala-hexarelin (0.33 nM) was incubated with rat cardiac membranes in the presence of increasing concentrations of GHRPs (1 nM to 0.1 mM) or of the endogenous ligands (inset) oxLDL (0.14–142 μg/ml) or TSP-1 (0.68–68 μg/ml) in a volume of 575 μl at 22 °C. Following UV irradiation, the covalently bound photoligand–CD36 complex was separated on SDS/PAGE, and bands corresponding to 88 kDa were counted for radioactivity. The IC₅₀ for oxLDL as a competitive ligand was 37 μM. The GHRPs used were hexarelin (□), GHRP-2 (▴), GHRP-6 (▵), EP80317 (▪), [D-Lys³]GHRP-6 (♦) and ghrelin (⋄). Results are means±S.E.M. from at least two independent experiments.

Figure 4. Effect of hexarelin (hex) on the dissociation of [¹²⁵I]oxLDL from HEK293/hCD36 cells

Cells were incubated for 2 h at 4 °C with 2.4 μg/ml [¹²⁵I]oxLDL. The cells were then washed and incubated with unlabelled oxLDL (300 μg/ml). At each time point (20, 40, 60, 90, 120 and 180 min), the cells were washed and cell-bound radioactivity was determined. The values are means±S.E.M. of triplicate determinations from one of two experiments giving similar results.

Figure 5. Electrophoretic properties of CNBr fragments of the [¹²⁵I]Tyr-Bpa-Ala-hexarelin–CD36 conjugate

(A) Rat heart CD36 was photolabelled with [¹²⁵I]Tyr-Bpa-Ala-hexarelin as described in the Materials and methods section (lane 1). After electrophoresis, the covalent photolabelled CD36 receptor was extracted from the gel and submitted to degradation with CNBr (100 mg/ml), yielding fragments of 30 and 2 kDa (lane 2). The deglycosylated ligand–receptor conjugate treated with CNBr yielded two fragments of 20 and 2 kDa (lane 3). The free radiolabelled photoactivable hexarelin derivative was loaded alone as a reference (lane 4). Electrophoresis was performed in a 16.5% (w/v) acrylamide gel. (B) Potential CNBr fragmentation pattern of CD36. The positions of the proteolytic fragments are indicated in square brackets. The molecular masses of the predicted photoaffinity cross-linked unglycosylated fragments and ligand are also indicated (kDa). The putative contact domains of [¹²⁵I]Tyr-Bpa-Ala-hexarelin, i.e. CD36-(Pro²⁸–Met¹⁶⁹) and CD36-(Ile²⁴⁶–Met⁴²⁹), are depicted with filled circles.

Figure 6. Electrophoretic properties of Endo Glu-C fragments of the [¹²⁵I]Tyr-Bpa-Ala-hexarelin–CD36 conjugate

(A) The band corresponding to the photolabelled CD36 receptor was extracted from the electrophoresis gel and submitted or not to a deglycosylation step. The 88 kDa receptor conjugate (glycosylated form) and its 55 kDa deglycosylated form were treated with Endo Glu-C (30 units for 72 h at 22 °C) and reloaded on a 16.5% (w/v) acrylamide gel. In both cases, a unique band corresponding to an 8 kDa fragment was obtained. (B) Potential Endo Glu-C fragmentation pattern of CD36. The fragment (∼8 kDa) resulting from Endo Glu-C digestion of the photolabelled receptor conjugate may correspond to the following modified fragments: CD36-(Gly⁴⁷–Glu⁷⁵), CD36-(Asn¹³²–Glu¹⁷⁷), CD36-(Val²⁸³–Glu³¹⁵) or CD36-(His³⁶⁷–Glu⁴⁰⁰) (depicted by filled circles).

Figure 7. CNBr cleavage of the partially purified Endo Glu-C- and Endo F-generated 8 kDa fragment of the photoligand–CD36 complex

(A) Autoradiography of the extracted band of ∼8 kDa obtained after treatment with Endo Glu-C (lane 1). Following CNBr treatment, two radioactive species (6 and 2 kDa) were obtained (lane 2). The free radioligand of 2 kDa was run in lane 3. (B) The proteolysis fragment (∼8 kDa) of the ligand–receptor complex obtained after treatment with Endo Glu-C and Endo-F, i.e. CD36(Asn¹³²–Glu¹⁷⁷), was subsequently digested with CNBr. After treatment with CNBr, two fragments of ∼6 and ∼2 kDa were obtained, the latter corresponding to the molecular mass of [¹²⁵I]Tyr-Bpa-Ala-hexarelin alone released from Met¹⁶⁹ (black circle).

Figure 8. Location of the binding site for [¹²⁵I]Tyr-Bpa-Ala-hexarelin on the primary sequence of CD36: comparison with the binding domains of CD36 for endogenous ligands

(A) Schematic summary of the fragmentation scheme employed with the photolabelled ligand–receptor conjugate. Digestion with Endo F, Endo Glu-C and CNBr was carried out as described in the Materials and methods section. The molecular masses of the fragment are indicated in kDa, and represent the actual sizes of the digested fragments including the ligand [¹²⁵I]Tyr-Bpa-Ala-hexarelin (hex). (B) Ligand-binding domains of CD36. The amino acid sequences of the binding domain for hexarelin, oxLDL and TSP-1 (endogenous ligands of CD36) are shown.