Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y12-like receptor members

Mutational analysis of gap junction formation.

The paired oocyte cell-cell channel assay was used to investigate the mechanisms involved in the process of formation of gap junction channels. Single oocytes, injected with connexin-specific mRNAs, accumulate a pool of precursors from which cell-cell channels can form rapidly upon pairing. Several lines of evidence, including immunohistochemistry and surface labeling, indicate that part of this precursor pool is located in the cell membrane, probably in the form of closed hemichannels. The homophilic binding of hemichannels to each other can be mimicked by synthetic peptides representing the extracellular loop sequences of connexin32. The peptides specifically suppress channel formation. A crucial role is established for the six cysteines in the extracellular domains that are conserved in all vertebrate gap junction proteins. Change of any of these cysteines into serines results in absolute loss of function of the mutant connexin. The effects of thiol-specific reagents on channel formation suggest that docking and/or opening of channels involves disulfide exchange. Several of the variable amino acids in the extracellular loop sequences were found to determine specificity of connexin-connexin interactions

Gating properties of connexin32 cell-cell channels and their mutants expressed in Xenopus oocytes

Carboxyl-terminal deletion mutants of the gap junction protein connexin32 were tested in the oocyte cell-cell channel assay. Oocytes expressing a mutant lacking 58 carboxyl terminal amino acids were found to exhibit junctional conductances of the same magnitude as oocytes expressing wild-type connexin32. The gating properties of the channels formed by this mutant of connexin32 with respect to transjunctional voltage and cytoplasmic acidification are indistinguishable from those found with wild-type connexin32 channels. This includes a novel pH-dependent voltage gate. In another mutant, two carboxyl terminal serine residues, Ser233 and Ser240, were replaced by Asn residues. This double mutant has properties indistinguishable from wild-type connexin32, suggesting that phosphorylation of either of these serines is not required for channel opening

Formation of hybrid cell-cell channels.

The oocyte cell-cell channel assay was used to demonstrate that connexin-43 is a cell-cell channel-forming protein as previously shown for connexin-32. Expression of connexin-32 in one and connexin-43 in the other oocyte of a pair results in the formation of junctional conductances at rates similar to those observed when only one or the other connexin is expressed in both oocytes of a pair. This suggests that hybrid cell-cell channels form in the oocyte system. Hybrid channels also form when a connexin-43 mRNA-injected oocyte is paired with a noninjected oocyte expressing endogenous connexin. The latter hybrids have properties apparently contributed by both types of hemichannels. Pure connexin-43 channels are not voltage gated, whereas pure oocyte channels are voltage dependent; hybrids of these channels rectify

Reduced activity of hypothalamic corticotropin-releasing hormone neurons in transgenic mice with impaired glucocorticoid receptor function.

Loss of central glucocorticoid receptor (GR) function is thought to be involved in the development of neuroendocrine and psychiatric disorders associated with corticotropin-releasing hormone (CRH) hyperactivity. The possible causal relationship between defective GR function and altered activity of CRH neurons was studied in transgenic mice (TG) expressing antisense RNA against GR. Immunocytochemical studies showed significant reductions in CRH immunoreactive neurons in the paraventricular nucleus (PVN) and in CRH and vasopressin (AVP) stores in the external zone of the median eminence. Concomitantly, stimulus-evoked CRH secretion from mediobasal hypothalami of TG micein vitrowas reduced significantly. However, CRH mRNA levels in the PVN of TG mice were marginally lower than those in wild-type (WT) mice.125I-CRH binding autoradiography revealed no differences between WT and TG animals in any of the brain regions that were studied. Basal plasma corticosterone (cort) levels and125I-CRH binding, CRH-R1mRNA, POMC mRNA, and POMC hnRNA levels in the anterior pituitary gland were similar in WT and TG mice. Intraperitoneal injection of interleukin-1β (IL-1β) increased plasma cort levels, CRH mRNA in the PVN, and anterior pituitary POMC hnRNA similarly in WT and TG mice. The injection of saline significantly reduced anterior pituitary CRH-R1mRNA levels in WT mice, but not in TG mice, whereas IL-1β produced a decrease in these mRNA levels in both strains.The data show that long-term GR dysfunction can be associated with reduced activity of CRH neurons in the PVN and decreased sensitivity of pituitary CRH-R1mRNA to stimulus-induced downregulation. Moreover, the hypothalamic changes observed in this model suggest that impaired GR function, at least if present since early embryonic life, does not necessarily result in CRH hyperexpression characteristics of disorders such as major depression.</jats:p

Anatomy of melancholia: focus on hypothalamic–pituitary–adrenal axis overactivity and the role of vasopressin

Overactivity of the hypothalamic–pituitary–adrenal (HPA) axis characterized by hypercortisolism, adrenal hyperplasia and abnormalities in negative feedback is the most consistently described biological abnormality in melancholic depression. Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are the main secretagogues of the HPA/stress system. Produced in the parvicellular division of the hypothalamic paraventricular nucleus the release of these peptides is influenced by inputs from monoaminergic neurones. In depression, anterior pituitary CRH1 receptors are down-regulated and response to CRH infusion is blunted. By contrast, vasopressin V3 receptors on the anterior pituitary show enhanced response to AVP stimulation and this enhancement plays a key role in maintaining HPA overactivity