The TLQP-21 peptide activates the G-protein-coupled receptor C3aR1 via a folding-upon-binding mechanism

Abstract

TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled receptor complement-3a receptor1 (C3aR1). The mechanisms of TLQP-21-induced receptor activation remain unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Additionally, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions.

Figure 1. TLQP-21 binding in 3T3L1 and CHO cells and activity at the C3aR1

A) Photoactivated crosslinking of a modified TLQP-21 peptide (CL) in CHO and 3T3 cells. CHO membranes were treated with PBS or CL (100 μM). A band of ~56 kDa was observed with anti-TLQP21 in membrane fractions P1and P2 from CL-treated but not PBS-treated membranes. Labeling remained in the protein pellet when P2 membranes were TCA-precipitated (P2 ppt). P2 membranes from 3T3 cells were treated with PBS or increasing concentrations of CL. B) Crosslinking of a modified TLQP-21 peptide (CL) to membranes prepared from 3T3 cells is reduced in the presence of the C3aR1 antagonist SB290157. Lanes 2 and 3 show that crosslinking is not affected by DMSO used to dissolve SB290157. C) The β-arrestin recruitment assay showed that TLQP-21 is an agonist for the human C3aR1, similarly to the human C3a peptide. The mouse TLQP-21 has higher potency than the human TLQP-21.

Figure 2. Structural analysis of TLQP-21 in presence of target 3T3L1 cells

Overlay spectra of refocused INEPT spectrum of TLQP-21 ¹³C and ¹⁵N labeled at P₄-R₆-A₉ (A,B), P₄-A₆-R₉-R₁₁-P₁₉-A₂₀ (C,D) or A₁₆-L₁₇-P₁₈ (E,F) with resonance assignments indicated. Experiments were performed in buffer (A,C,E) or in the presence of 3T3L1 cells after 24 hour incubation at 37°C (B,D,F). G,H. Chemical shift index of TLQP-21 in buffer (red) or in the presence of 3T3L1 cells (blue) (Schwarzinger, et al., 2000).

Figure 3. A structural model of TLQP-21 upon receptor binding

The backbone conformation was obtained with XPLOR-NIH software (Schwieters et al., 2003) constraining the dihedral angles of the residues showing helical chemical shifts. The conformation of the side chains is arbitrary. Electrostatic surface calculated with APBS (Baker et al., 2001) illustrates the positively charged N-terminal domain of TLQP-21 and primarily neutral C-terminal domain.

Figure 4. Structural analysis of TLQP-21 in presence of C3aR1 knockout cells and a C3aR1 antagonist

Refocused INEPT spectra of TLQP-21, labeled with ¹³C and ¹⁵N at P₄-A₆-R₉ with resonance assignments indicated. Experiments were performed in buffer (A,D), in the presence of 3T3L1 cells (B), in the presence of 3T3L1 cells incubated with the C3aR1 antagonist SB290157 (C), in the presence of splenocytes derived from wild type (E) or in the presence of splenocytes derived from a C3aR1 knockout mice (F).

Figure 5. TLQP-21 but not R21A mutant enhances lipolysis in 3T3L1

A) TLQP-21 potentiates lipolysis induced by the β-adrenergic receptor agonist isoproterenol (ISO) (F(5,65)=62.1, p<0.0001). B) Unlike TLQP-21, the R21A mutant peptide does not potentiate isoproterenol-induced lipolysis in 3T3L1 adipocytes (F(5,65)=33.4, p<0.00001). C) Refocused INEPT spectra of R21A mutant, labeled with ¹³C and ¹⁵N at A₁₆-L₁₇-P₁₈ with resonance assignments indicated. Experiments were performed in buffer (left panel) or in the presence of 3T3L1 cells (right panel). *p<0.05, ** p<0.001.