Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A1 and P2Y2 receptors in rat brain

<p>Abstract</p> <p>Background</p> <p>Purines such as adenosine and ATP are now generally recognized as the regulators of many physiological functions, such as neurotransmission, pain, cardiac function, and immune responses. Purines exert their functions via purinergic receptors, which are divided into adenosine and P2 receptors. Recently, we demonstrated that the G_i/o-coupled adenosine A₁receptor (A₁R) and G_q/11-coupled P2Y₂receptor (P2Y₂R) form a heteromeric complex with unique pharmacology in co-transfected human embryonic kidney cells (HEK293T). However, the heteromeric interaction of A₁R and P2Y₂R <it>in situ </it>in brain is still largely unknown.</p> <p>Findings</p> <p>In the present study, we visualized the surface expression and co-localization of A₁R and P2Y₂R in both transfected HEK293T cells and in rat brain by confocal microscopy and more precisely by immunogold electron microscopy. Immunogold electron microscopy showed the evidence for the existence of homo- and hetero-dimers among A₁R and P2Y₂R at the neurons in cortex, cerebellum, and particularly cerebellar Purkinje cells, also supported by co-immunoprecipitation study.</p> <p>Conclusion</p> <p>The results suggest that evidence for the existence of homo- and hetero-dimers of A₁R and P2Y₂R, not only in co-transfected cultured cells, but also <it>in situ </it>on the surface of neurons in various brain regions. While the homo-dimerization ratios displayed similar patterns in all three regions, the rates of hetero-dimerization were prominent in hippocampal pyramidal cells among the three regions.</p

Regulation of GIP and GLP1 Receptor Cell Surface Expression by N-Glycosylation and Receptor Heteromerization

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer

Specific oligomerization of the 5-HT1A receptor in the plasma membrane

In the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Förster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between CFP-and YFP-tagged 5-HT1A receptors at the plasma membrane. Through the analysis of the level of apparent FRET efficiency over the various relative amounts of donor and acceptor, as well as over a range of total surface expressions of the receptor, we verify the specific interaction of these receptors. Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT1A receptor mutant. Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane. This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction

Circadian-Related Heteromerization of Adrenergic and Dopamine D4 Receptors Modulates Melatonin Synthesis and Release in the Pineal Gland

Dopamine and adrenergic receptor complexes form under a circadian-regulated cycle and directly modulate melatonin synthesis and release from the pineal gland

Circadian-Related Heteromerization of Adrenergic and Dopamine D4 Receptors Modulates Melatonin Synthesis and Release in the Pineal Gland

Dopamine and adrenergic receptor complexes form under a circadian-regulated cycle and directly modulate melatonin synthesis and release from the pineal gland

Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

A single-molecule tracking technique coupled with mathematical modeling was developed for fully determining the dynamic monomer–dimer equilibrium of molecules in or on the plasma membrane, which will provide a framework for understanding signal transduction pathways initiated and regulated by dynamic dimers of membrane-localized receptors

BRET analysis of GPCR oligomerization: newer does not mean better

International audienceN.A

CXCR4–CCR5: A couple modulating T cell functions

Chemokines and their receptors direct leukocyte migration among blood, lymph and tissues. Evidence has recently accumulated that, besides their chemotactic functions, chemokine receptors are highly versatile players that fine tune immune responses. During human T cell activation by antigen-presenting cells, the chemokine receptors CCR5 and CXCR4 are recruited into the immunological synapse, where they deliver costimulatory signals. However, the molecular mechanisms allowing signaling versatility of chemokine receptors are unknown. Here, we describe the functional interaction between CXCR4 and CCR5 to exert specific biological functions and modulate T lymphocyte responses. We demonstrate that simultaneous expression and cooperation between CCR5 and CXCR4 are required for chemokine-induced T cell costimulation at the immunological synapse. In addition, we provide evidence for a physical association of the two receptors in a signaling complex that activates distinct T cell functions. We suggest that cooperation between receptors represents one key strategy for the functional plasticity of chemokines