Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. Photoaffinity, NMR, and molecular modeling

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors named VIP pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPACs). The molecular nature of VIP binding to receptors remains elusive. In this work, we have docked VIP in the human VPAC1 receptor by the following approach. (i) VIP probes containing photolabile residues in positions 6, 22, and 24 of VIP were used to photolabel the receptor. After receptor cleavage and Edman sequencing of labeled receptor fragments, it was shown that Phe6, Tyr22, and Asn24 of VIP are in contact with Asp107, Gly116, and Cys122 in the N-terminal ectodomain (N-ted) of the receptor, respectively. (ii) The structure of VIP was determined by NMR showing a central alpha helix, a disordered N-terminal His1-Phe6 segment and a 3(10) Ser25-Asn28 helix termination. (iii) A three-dimensional model of the N-ted of hVPAC1 was constructed by using the NMR structure of the N-ted of corticotropin-releasing factor receptor 2beta as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel beta sheets and is stabilized by three disulfide bonds. (iv) Taking into account the constraints provided by photoaffinity, VIP was docked into the hVPAC1 receptor N-ted. The 6-28 fragment of VIP nicely lies in the N-ted C-terminal part, but the N terminus region of VIP is free for interacting with the receptor transmembrane region. The data provide a structural rationale to the proposed two-step activation mechanism of VPAC receptor and more generally of class II G protein-coupled receptors.     

Purinergic Receptors Crosstalk with CCR5 to Amplify Ca2+ Signaling

Cellular and Molecular Neurobiology - Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C–C chemokine receptor type...     

The vasoactive intestinal peptide (VIP) alpha-Helix up to C terminus interacts with the N-terminal ectodomain of the human VIP/Pituitary adenylate cyclase-activating peptide 1 receptor: photoaffinity, molecular modeling, and dynamics

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors. We characterized the C terminus-binding site of VIP in the N-terminal ectodomain (N-ted) of the human VPAC1 receptor: 1) The probe [(125)I-Bpa(28)]VIP in which the C-terminal residue (Asn(28)) is substituted by a photoreactive p-benzoyl-l-Phe (Bpa) was used to photolabel the receptor. After receptor cleavage and Edman sequencing, it was shown that Asn(28) of VIP is in contact with Lys(127) in the receptor N-ted. Taking into account previous data, it follows that the C-terminal and central parts of VIP from Asn(28) to Phe(6) lie in the N-ted. 2) A three-dimensional model of the N-ted was constructed, the fold being identified as a Sushi domain with two antiparallel beta-sheets and three disulfide bonds. The nuclear magnetic resonance structure of VIP was then docked into this model by taking into account the constraint provided by photoaffinity experiments with [(125)I-Bpa(28)]VIP. It appeared that VIP runs parallel to the beta3-beta4 antiparallel sheets. 3) We performed molecular dynamic simulations over 14 nsec of the complex between VIP and receptor N-ted and the free N-ted. The structural model of the free N-ted is stable, and VIP tends to further stabilize the N-ted structure more especially in the loops connecting the beta-sheets. These structural studies provide a detailed molecular understanding of the VIP-receptor interaction.     

Biased Signaling Favoring Gi over β-Arrestin Promoted by an Apelin Fragment Lacking the C-terminal Phenylalanine

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or G_i protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a G_i-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates G_i and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its G_i activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely G_i-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the G_i-dependent signaling may participate in K17F-induced BP decrease.     

Placental hCG immunohistochemistry and serum free-Beta-hCG at 11–13 weeks’ gestation in intrauterine fetal demise

Intrauterine fetal demise (IUFD) is a continuing problem that can result in severe psychosocial trauma for expecting parents. Our aim was to analyze placental human chorionic gonadotropin (hCG) expression at the third trimester and free-Beta-hCG levels measured at 11–13 weeks in cases of IUFD that occurred after 34 weeks’ gestation, alongside a parallel analysis of a set of controls. In this retrospective study we present immunohistochemical data of a tissue microarray that included the following: 12 placentas where IUFD occurred (24 samples); 28 control placentas from first and early second trimester (56 samples); and 30 control placentas at term of pregnancy (60 samples). We used immunohistochemistry to analyze the expression of hCG. Data are also presented from 3,240 first trimester trisomies screening tests, of which 21 pregnancies resulted in IUFD (15 after 22 weeks’ gestation and 6 after 34 weeks). All pregnancies took place between 2001 and 2010. For each case, our analysis took account of pregnancy-related data that we gathered from the relevant clinical files. Small for gestational age (SGA) was defined as neonatal weight <10th centile. Our results show that full-term placentas displayed a decreased immunohistochemical expression of hCG in comparison with those at the first trimester (p < 0.05). Moreover, low hCG expression in placentas at the third trimester was shown to be an independent risk factor for IUFD after 34 weeks’ gestation (under multivariate analysis with p < 0.05). When we reviewed first trimester screening results, free-Beta-HCG was found to be lower for the group of IUFD after 34 weeks’ gestation than in the group of live births (p < 0.05). This difference was heavily weighted by non small for gestational age (non-SGA) associated cases of IUFD: these presented a free-Beta-hCG MoM log of ?0.27 (±0.09) in contrast to just ?0.01 (±0.03) in SGA-associated IUFD (p < 0.05). Our results show that low hCG is an independent risk factor for IUFD after 34 weeks’ gestation, and that levels of the hormone are significantly lower in non-SGA associated cases of IUFD.