Francesca Guzzi

Localization of the human oxytocin receptor in caveolin-1 enriched domains turns the receptor-mediated inhibition of cell growth into a proliferative response.

In this study, we investigated the functional role of the localization of human OTR in caveolin-1 enriched membrane domains. Biochemical fractionation of MDCK cells stably expressing the WT OTR-GFP indicated that only minor quantities of receptor are partitioned in caveolin-1 enriched domains. However, when fused to caveolin-2, the OTR protein proved to be exclusively localized in caveolin-1 enriched fractions, where it bound the agonist with increased affinity and efficiently coupled to Gαq/11. Interestingly, the chimeric protein was unable to undergo agonist-induced internalization and remained confined to the plasma membrane even after prolonged agonist exposure (120 min). A striking difference in receptor stimulation was observed when the OT-induced effect on cell proliferation was analysed: stimulation of the human WT OTR inhibited cell growth, whereas the chimeric protein had a proliferative effect. These data indicate that the localization of human OTR in caveolin-1 enriched microdomains radically alters its regulatory effects on cell growth; the fraction of OTR residing in caveolar structures may therefore play a crucial role in regulating cell proliferation.

Sphingolipid Metabolism and Caveolin Expression in Gonadotropin-Releasing Hormone-Expressing GN11 and Gonadotropin-Releasing Hormone-Secreting GT1-7 Neuronal Cells

In this paper, we show that caveolin-1 is abundantly present in a cell line of immortalized gonadotropin-releasing hormone-expressing neurons (GN11). In contrast to GN11, caveolin is undetectable in a cognate cell line of immortalized gonadotropin-releasing hormone-secreting neurons (GT1-7). These two cell lines are characterized by a radically different sphingolipid metabolism. After incubation in the presence of tracer amount of [1-3H]sphingosine, GN11 and GT1-7 neurons incorporated similar amounts of radioactivity. In GT1-7 neurons, [1-3H]sphingosine metabolism was markedly oriented toward the biosynthesis of complex sphingolipids. In fact, almost all the radioactivity in the lipid extracts from GT1-7 cells was associated with biosynthetic products (ceramide, sphingomyelin, and glycosphingolipids). In particular glycosphingolipids represented more than 65% of total lipid radioactivity in these cells, and the main glycosphingolipid was GM3 ganglioside (about 47% of total lipid radioactivity). In the case of GN11 neurons, a high portion of [1-3H]sphingosine underwent complete degradation, as indicated by the formation of high levels of radioactive phosphatidylethanolamine (about 23% of lipid radioactivity). Moreover, the main complex sphingolipid in GN11 neurons was not a glycolipid, but sphingomyelin (its level in these cells, about 54% of lipid radioactivity, was two-fold higher than in GT1-7). Glycolipids, gangliosides in particular, were present in low amount (9.5% of lipid radioactivity) if compared with the cognate GT1-7 cell line, and GM3 was almost absent in GN11 neurons. Despite the radical differences in ganglioside and caveolin content, from both cell types a membrane fraction similarly enriched in sphingolipids was prepared. In the case of GN11 cells, this fraction was also enriched in caveolin. The presence of caveolin or GM3 may correlate with different functional properties linked to the stage of neuronal maturation, since GN11 and GT1-7 are representative, respectively, of immature, migrating, and differentiated, postmigratory gonadotropin-releasing hormone-positive neurons.

Light on the structure of thromboxane A2 receptor heterodimers

The structure-based design of a mutant form of the thromboxane A2 prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the hetero-dimeric complexes between the TPα and β isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPα–TPβ hetero-dimerization serves to regulate TPα function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution.

Polarization of caveolins and caveolae during migration of immortalized neurons.

During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin‐releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl‐β‐cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin‐1 isoform (cav‐1) in the leading processes. In contrast, during two‐dimensional migration cav‐1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.

Presence of delta opioid receptors on a subset of hypothalamic gonadotropin releasing hormone (GnRH) neurons

Opioid peptides exert an inhibitory effect on hypothalamic gonadotropin releasing hormone (GnRH) secretion mainly by interacting with mu-opioid receptors. Although a direct role for opioids via delta-opioid receptors (DORs) has been suggested, the presence of these receptors on GnRH neurons has never been demonstrated. In the present study, we determined the distribution of DORs in the basal hypothalamus of rat with special focus on their relation to GnRH neurons. Double-labelling immunofluorescence and confocal microscopy revealed that DORs are exclusively present in a subpopulation of GnRH nerve terminals, with the highest density in the external layer of the median eminence. We then studied the functional characteristics of DORs in an immortalized GnRH-secreting neuronal cell line (GT1-1) known to endogenously express this receptor. Here, pertussis toxin pretreatment abolished the delta-agonist (DPDPE) inhibitory effect on cAMP accumulation. We also analyzed the type of G proteins involved in the signal transduced by the DOR and showed that GT1-1 cells express the inhibitory Go and Gi2 alpha-subunits. However, only Go was down-regulated under chronic DPDPE exposure. Finally, since DOR is expressed postnatally in brain, we compared GnRH neuronal cells immortalized at different developmental stages (the more mature GT1-1 and GT1-7 cells, versus the more immature GN11 cells), evidencing that only mature neurons express DOR. In conclusion, our study indicates that a direct control of opioids via delta-receptors occurs on GnRH neurons and validates the use of GT1 cells to further investigate the nature of the DOR present on GnRH neurons.

Thioacylation is required for targeting G-protein subunit G(o1alpha) to detergent-insoluble caveolin-containing membrane domains.

alpha-Subunits of heterotrimeric G(i)-like proteins (alpha(i), alpha(o) and alpha(z)) associate with the cytoplasmic leaflet of the plasma membrane by means of N-terminally linked myristic acid and palmitic acid. An additional role for palmitate has been recently suggested by the observation that fusion with the palmitoylated N-terminus of alpha(i1) relocalizes cytosolic green-fluorescent-protein reporter to low buoyancy, Triton-insoluble membrane domains (TIFF; Triton-insoluble floating fraction), enriched with caveolin-1 [Galbiati, Volonté, Meani, Milligan, Lublin, Lisanti and Parenti (1999) J. Biol. Chem 274, 5843-5850]. Here we show that, upon transient expression in transfected COS-7 cells, myristoylated and palmitoylated alpha(o) (alpha(o)wt, where wt is wild-type) is exclusively found in TIFF, from where non-palmitoylated alpha(o)wt and alpha(o)C3S (Cys(3)-->Ser) mutant are excluded. Moreover, alpha(o) fused to N-terminally truncated human vasopressin V2 receptor (V2TR-alpha(o)), lacking myristate and palmitate, still localizes at the plasma membrane by means of first transmembrane helix of V2R, but is excluded from TIFF. Likewise, alpha(o)C3S does not partition into TIFF, even when its membrane avidity is enhanced by co-expression of betagamma-subunits. Thus membrane association, in the absence of added palmitate, is not sufficient to confer partitioning of alpha(o) within TIFF, suggesting that palmitoylation is a signal for membrane compartmentalization of dually acylated alpha-subunits.

Palmitic Is the Main Fatty Acid Carried by Lipids of Detergent-Resistant Membrane Fractions from Neural and Non-Neural Cells

Lipids extracted from detergent-resistant membrane fractions, thought to derive from membrane domains, were analyzed for fatty acid composition. The proportion of palmitic acid in fractions isolated from neurons (cerebellar granule cells) and from neural-like cell lines (neuroblastoma-glioma NG108-15) nearly doubled (reaching about 54% of total fatty acids) with respect to cell WCL, indicating their enrichment in palmitic acid-carrying lipids. The proportion of palmitic acid in detergent-resistant fractions obtained from caveolin-transfected NG108-15 cells was comparable with that obtained from caveolin-negative cells, ruling out a specific role of this protein in recruiting palmitoylated lipid species. The enrichment in palmitic acid was remarked also in membrane fractions isolated from non-neuronal cell lines (A431) using either detergents or detergent-free techniques. Lipid fractionation and mass spectrometry experiments show that palmitic acid–rich phosphatidylcholine species are responsible of the peculiar fatty acid composition of these fractions. All together these results suggest that the enrichment in palmitic acid–rich phosphatidylcholine species is a common feature of neural and non-neural cell lines and may play a major role in the biogenesis of membrane domains.

Heterotrimeric G proteins demonstrate differential sensitivity to β-arrestin dependent desensitization

G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization. Enhancing beta2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and delta opioid receptors. Furthermore, co-immunoprecipitation experiments showed that G15 alpha subunit (as opposed to Galphaq and Galphas) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by beta-arrestin. Interestingly, co-expression of Galpha15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced beta2 adrenergic receptor internalization. Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters.

Retinoic acid- and phorbol ester-induced neuronal differentiation down-regulates caveolin expression in GnRH neurons

J. Neurochem. (2008) 104, 1577–1587.

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo

Graphical abstract Figure. No caption available. ABSTRACT Thromboxane A2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein‐coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TP&agr; and TP&bgr; homo‐/hetero‐dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss‐of‐function TP&agr; variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK‐293T cells. These findings provide proofs that lack of homo‐dimer formation is a crucial process for reduced TP&agr; function in vivo, and might represent one molecular mechanism through which platelet TP&agr; receptor dysfunction affects the patient(s) carrying these mutations.