Search

Summary: G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry. Notably, we detail a label-free quantitation strategy that eliminates irrelevant interacting proteins using an inactive GPR110 mutant with impaired surface expression. Furthermore, we outline procedures for validating the identified partners.For complete details on the use and execution of this protocol, please refer to Huang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Activation of adhesion receptor GPR110 by the endogenous ligand synaptamide promotes neurogenesis, neurite growth, and synaptogenesis in developing brains through cAMP signal transduction. However, interacting partners of GPR110 and their involvement in cellular function remain unclear. Here, we demonstrate using chemical crosslinking, affinity purification, and quantitative mass spectrometry that GPR110 interacts with the tight junction adhesion protein occludin. By removing non-specific partners by comparing the binding proteins of GPR110 WT and an inactive mutant exhibiting impaired surface expression, occludin was distinguished as a true binding partner which was further confirmed by reciprocal co-immunoprecipitation assay. Deletion of GPR110 in mice led to the disruption of blood-brain barrier (BBB) and reduced occludin phosphorylation at Y285 in the brain. The Y285 phosphorylation increased upon the ligand-induced activation of GPR110. These data suggest an important role of GPR110-occludin interaction in BBB function and association of previously unknown GPR110-dependent occludin phosphorylation at Y285 with BBB integrity.

Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research.

Phosphatidylserine (PS), the major anionic phospholipid in eukaryotic cell membranes, is synthesized by the integral membrane enzymes PS synthase 1 (PSS1) and 2 (PSS2). PSS2 is highly expressed in specific tissues, such as brain and testis, where docosahexaenoic acid (DHA, 22:6n-3) is also highly enriched. The purpose of this work was to characterize the hydrocarbon-chain preference of PSS2 to gain insight on the specialized role of PSS2 in PS accumulation in the DHA-abundant tissues. Flag-tagged PSS2 was expressed in HEK cells and immunopurified in a functionally active form. Purified PSS2 utilized both PE plasmalogen and diacyl PE as substrates. Nevertheless, the latter was six times better utilized, indicating the importance of an ester linkage at the sn-1 position. Although no sn-1 fatty acyl preference was noted, PSS2 exhibited significant preference toward DHA compared with 18:1 or 20:4 at the sn-2 position. Preferential production of DHA-containing PS (DHA-PS) was consistently observed with PSS2 purified from a variety of cell lines as well as with microsomes from mutant cells in which PS synthesis relies primarily on PSS2. These findings suggest that PSS2 may play a key role in PS accumulation in brain and testis through high activity toward DHA-containing substrates that are abundant in these tissues.

In this study, we have examined the effects of n-3 fatty acid deficient diets on the phospholipids (PL) molecular species composition in the hippocampus. Female rats were raised for two generations on diets containing linoleic acid (18:2n-6), with or without supplementation of α-linolenic acid (18:3n-3) or 18:3n-3 plus docosahexaenoic acid (22:6n-3). At 84 days of age, the hippocampal phospholipids were analyzed by reversed phase HPLC-electrospray ionization mass spectrometry. Depleting n-3 fatty acids from the diet led to a reduction of 22:6n-3 molecular species in phosphatidylcholine (PC), phosphatidylethanolamine (PE), PE-plasmalogens (PLE), and phosphatidylserine (PS) by 70–80%. In general, 22:6n-3 was replaced with 22:5n-6 but the replacement at the molecular species level did not always occur in a reciprocal manner, especially in PC and PLE. In PC, the 16:0,22:6n-3 species was replaced by 16:0,22:5n-6 and 18:0,22:5n-6. In PLE, substantial increases of both 22:5n-6 and 22:4n-6 species compensated for the decreases in 22:6n-3 species in n-3 fatty acid deficient groups. While the total PL content was not affected by n-3 deficiency, the relative distribution of PS decreased by 28% with a concomitant increase in PC. The observed decrease of 22:6n-3 species along with PS reduction may represent key biochemical changes underlying losses in brain-hippocampal function associated with n-3 deficiency.—Murthy, M., J. Hamilton, R. S. Greiner, T. Moriguchi, N. Salem, Jr., and H-Y. Kim. Differential effects of n-3 fatty acid deficiency on phospholipid molecular species composition in the rat hippocampus. J. Lipid Res. 2002. 43: 611–617.

N–3 essential fatty acid deficiency affects a number of biological and physiological processes. In this study, we investigated the effect of n–3 essential fatty acid status on two key pineal biochemical functions, melatonin production and lipoxygenation, using pineal glands from rats given an n–3-adequate or n–3-deficient diet. The pineal total lipid profile and phospholipid molecular species distribution altered by n–3 deficiency were evaluated in parallel. In pineal glands from n–3-deficient rats, an 87% reduction of 22:6n–3 (docosahexaenoic acid) was observed, and this decrease was accompanied by increases in 22:4n–6 (docosatetraenoic acid, 3-fold), 22:5n–6 (docosapentaenoic acid, 12-fold), and 20:4n–6 (arachidonic acid, 48%). The significant decrease of 22:6n–3 containing species in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) was also evident. These decreases in 22:6n–3 containing PL species were compensated by substantial accumulations of 22:4n–6 or 22:5n–6 and slight increases in 20:4n–6 containing PL species in PC and PE. In PS, however, the accumulation of n-6 species was not adequate to compensate for the loss of 22:6n–3 species. N–3 deficiency significantly reduced non-esterified 20:4n–6 and 22:6n–3 levels in pineals (25% and 65%, respectively). Concomitantly, the endogenous 12-HETE level decreased by 35% in deficient pineals. In contrast, n–3 deficiency led to a more than 60% increase in the daytime pineal melatonin level. In conclusion, n–3 fatty acid deficiency not only has profound effects on pineal lipid profiles but also on pineal biochemical activities. These results suggest that n–3 fatty acids may play a critical role in regulating pineal function.—Zhang, H., J. H. Hamilton, N. Salem, Jr., and H-Y. Kim. N–3 fatty acid deficiency in the rat pineal gland: effects on phospholipid molecular species composition and endogenous levels of melatonin and lipoxygenase products. J. Lipid Res. 1998. 39: 1397–1403.