Aljoša Bavec

Different role of intracellular loops of glucagon-like peptide-1 receptor in G-protein coupling

Previous studies revealed the importance of the third intracellular loop of glucagon-like peptide-1 receptor (GLP-1R) in coupling to G(s) and G(i1) proteins. In order to further study the signaling mechanisms of GLP-1R, we tested three peptides, corresponding to the sequences of the first (IC(1)), the second (IC(2)), and the third (IC(3)) intracellular loop of GLP-1R, for their interactions with heterotrimeric G-proteins of different types (G(alphas), G(alphao), G(alphai1), and G(alpha11) plus G(beta1gamma2)) overexpressed in sf9 cells. IC(3) peptide powerfully stimulates all types of tested G-proteins, whereas IC(1) and IC(2) peptides show differential effects on G-proteins. Both IC(1) and IC(2) peptides activate G(s) and cooperate with IC(3) peptide in its stimulation. G(o) is not affected by IC(1) and IC(2). G(i1) and G(11) are not affected by IC(1), but are activated by IC(2), which in activation cooperates with IC(3). We suggest that GLP-1R is not coupled only to G(s) and G(i1), as shown previously, but also to G(o) and G(11). IC(3) loop is the main switch that mediates signaling via GLP-1R to G-proteins, while IC(1) and IC(2) loops are important in discrimination between different types of G-proteins.

PEPTITERGENT PD1 AFFECTS THE GTPASE ACTIVITY OF RAT BRAIN CORTICAL MEMBRANES

Peptitergent PD1 shows complex effects on GTPase activity of rat brain cortical membranes: inhibition in the presence of lower concentrations of GTP and activation at a higher concentration, above 0.5 microM, of GTP. Its effect is dose dependent and is characterized by an EC50 of 1.8 +/- 0.2 microM and a Hill coefficient of 1.6 +/- 0.3, and it increases both Km and Vmax of the GTP hydrolysis. PD1 that was unable to solubilize G-proteins from the membranes probably acts on them by direct binding near the C-terminal alpha-helical region of the Galpha subunit, similarly to mastoparan.

Cell-Penetrating Mimics of Agonist-Activated G-Protein Coupled Receptors

Cell-penetrating peptides have proven themselves as valuable vectors for intracellular delivery. Relatively little is known about the frequency of cell-penetrating sequences in native proteins and their functional role. By computational comparison of peptide sequences, we recently predicted that intracellular loops of G-protein coupled receptors (GPCR) have high probability for occurrence of cell-penetrating motifs. Since the loops are also receptor and G-protein interaction sites, we postulated that the short cell-penetrating peptides, derived from GPCR, when applied extracellularly can pass the membrane and modulate G-protein activity similarly to parent receptor proteins. Two model systems were analyzed as proofs of the principle. A peptide based on the C-terminal intracellular sequence of the rat angiotensin receptor (AT1AR) is shown to internalize into live cells and elicit blood vessel contraction even in the presence of AT1AR antagonist Sar1-Thr8-angiotensin II. The peptide interacts with the same selectivity towards G-protein subtypes as agonist-activated AT1AR and blockade of phospholipase C abolishes its effect. Another cell-penetrating peptide, G53-2 derived from human glucagon-like peptide receptor (GLP-1R) is shown to induce insulin release from isolated pancreatic islets. The mechanism was again found to be shared with the original GLP-1R, namely G11-mediated inositol 1,4,5-triphosphate release pathway. These data reveal a novel possibility to mimic the effects of signalling transmembrane proteins by application of shorter peptide fragments.

Immunoassay for visualization of protein‐protein interactions on Ni‐nitrilotriacetate support: Example of a laboratory exercise with recombinant heterotrimeric Gαi2β1γ2 Tagged by Hexahistidine from sf9 Cells

We have developed an in vitro assay for following the interaction between the αi2 subunit and β1γ2 dimer from sf9 cells. This method is suitable for education purposes because it is easy, reliable, nonexpensive, can be applied for a big class of 20 students, and avoid the commonly used kinetic approach, which does not allow visualization of protein‐protein interaction.

Functional characterization of N-terminally GFP-tagged GLP-1 receptor.

The glucagon-like peptide-1 receptor (GLP-1 receptor) mediates important effects on peripheral tissues and the central nervous system. It seems one of the most promising therapeutic targets for treatment of diabetes mellitus type 2. Surprisingly, very little is known about the cellular mechanisms that regulate its function in vivo. One of the approaches to study receptor dynamics, expression, or signaling is using GFP-tagged fluorescent proteins. In this study, we synthesized and characterized N-terminally GFP-tagged GLP-1 (GFP-GLP-1) receptor in CHO cells. We demonstrated that GFP-GLP-1 receptor is weakly expressed in the plasma membranes and is functionally coupled to adenylyl cyclase via heterotrimeric G-proteins, similarly as its wild type.

Constructing glucagon like peptide-1 receptor fused with derivatives of GFP for visualizing protein–protein interaction in living cells

The glucagon-like peptide-1 receptor (GLP-1 receptor) mediates important antidiabetogenic effects on peripheral tissues. It appears to be one of the most promising therapeutic targets for treatment of diabetes mellitus type 2. Surprisingly, very little is known about the cellular mechanisms that regulate receptor function in living cells. One of the approaches how to study receptor dynamics is by using tagged fluorescent proteins. In this study, YFP-tagged GLP-1 (YFP-GLP-1) receptor and CFP-tagged GLP-1 (CFP-GLP-1) receptor for visualizing protein–protein interaction in living cells were constructed and localized in CHO cells. Cells expressing YFP-GLP-1 and CFP-GLP-1 receptor showed characteristic GLP-1 mediated increase in cAMP, similar to cells expressing a wild type GLP-1 receptor. This means that both types of receptors are functional and localized in plasma membrane.

Role of cysteine 341 and arginine 348 of GLP-1 receptor in G-protein coupling

We have demonstrated the ability of peptides derived from the third intracellular loop of GLP-1 receptor to differently modulate activity of four different types of G-proteins overexpressed in sf9 cells. In this respect, the involvement of Cys341 in inhibition of Gs and Cys341 in activation of Gs and in inhibition of Gi1, Go, and G11, respectively, indicates their potential role in discrimination between different types of G-proteins. Moreover, these two amino acids from the third intracellular loop might represent an important novel targets for covalent modification by downstream regulators in signaling through GLP-1 receptor.

Third intracellular loop of glucagon like-peptide-1 receptor is coupled with endogenous mono-ADP-ribosyltransferase — Novel type of receptor regulation?

Our previous studies revealed the main role of the third intracellular loop (IC(3)) of glucagon-like peptide-1 receptor (GLP-1 receptor), in G-protein activation, where the presence or absence of agonist and the receptor phosphorylation seemed to be the only regulatory mechanisms. In order to further study the signaling mechanisms of GLP-1 receptor, we investigated the effect of the third intracellular loop-derived peptide on endogenous mono-ADP-ribosyltransferase mediated mono-ADP-ribosylation of G-proteins β subunit in CHO cells. Results showed an inhibitory effect of IC(3) peptide on mono-ADP-ribosylation of β subunit, obviously via the mechanism of competitive inhibition. Excluding the activity of this inhibitory mechanism via pertussis toxin-sensitive G proteins, the direct functional coupling of IC(3) of GLP-1 receptor and endogenous mono-ADP-ribosyltransferase was confirmed. We suggest that this arginine specific enzymatic posttranslational modification of third intracellular loop of GLP-1 receptor might represent a possible novel mechanism of receptor activity regulation and the pharmacological potential in treatment of diabetes mellitus type 2.

G-Protein coupled progesterone receptors in the plasma membrane of fungus Rhizopus nigricans

Abstract We have demonstrated simultaneous existence of progesterone receptors and GTPase activity in the membranes prepared from the filamentous fungus Rhizopus nigricans. The results obtained with pertussis toxin treated fungal mycelium suggest that these receptors do not couple to Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.We have demonstrated simultaneous existence of progesterone receptors and GTPase activity in the membranes prepared from the filamentous fungus Rhizopus nigricans. The results obtained with pertussis toxin treated fungal mycelium suggest that these receptors do not couple to Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.

(Poly)peptide-based therapy for diabetes mellitus: insulins versus incretins.

Insulin therapy remains the standard of care for achieving and maintaining adequate glycemic control, especially in hospitalized patients with critical and noncritical illnesses. Insulin therapy is more effective against elevated fasting glycaemia but less in the reduction of postprandial hyperglycaemia. It is associated with a high incidence of hypoglycemia and weight gain. Contrary, GLP-1 mimetic therapy improves postprandial glycaemia without the hypoglycaemia and weight gain associated with aggressive insulin therapy. Moreover, it has the potential to reduce cardiovascular related morbidity. However, its increased immunogenicity and severe gastrointestinal adverse effects present a huge burden on patients. Thus, a right combination of basal insulin which has lowering effect on fasting plasma glucose and GLP-1 mimetic with its lowering effect on postprandial plasma glucose with minimal gastrointestinal adverse effects, seems the right therapy choice from a clinical point of view for some diabetic patients. In this article, we discuss the pros and cons of the use of insulin analogues and GLP-1 mimetics that are associated with the treatment of type 2 diabetes.