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Neuropeptide Y (NPY) is universally expressed in many different neuronal and non-neuronal cells. Human NPY gene has two in-frame kozak sequences and thus, has potentially two translation initiation sites producing two NPY peptides with different molecular weights. In the present study, the intracellular location of NPY was studied in endothelial cells endogenously expressing NPY, and in neuronal (SK-N-BE) and non-neuronal (CHO-K1) cells transfected with NPY-GFP-constructs. By mutating kozak sequences we discovered that kozak-1 directs the NPY peptide to secretory vesicles, and kozak-2 is a prerequisite for mitochondrial targeting. If both kozak sequences are present, non-neuronal cells seem to benefit leaky scanning to initiate translation at both initiation sites, in contrast to neuronal cells, which prefer the kozak-1. This finding suggests that both the kozak sequences of NPY mRNA can be used in the translation depending on the cell type. The size and the function of the novel NPY fragment routed to mitochondria remains to be determined.

Most G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation followed by arrestin binding and internalization. In this study we explored the time-, ligand-, and concentration dependence of alpha2-adrenoceptor internalization in human embryonal kidney (HEK-293) cells expressing alpha2A- and alpha2B-adrenoceptors. We also explored the relationship between ligand-induced receptor internalization and agonist efficacy, determined with a [35S]GTPgammaS binding assay. The results showed rapid dose-dependent internalization of both alpha2A- and alpha2B-receptors; the extent of internalization was directly proportional to agonist efficacy. The agonist UK 14,304 had a subtype-specific high efficacy at alpha2A-AR and dexmedetomidine at alpha2B-AR. Agonist-induced [35S]GTPgammaS binding was totally blocked by pretreatment with pertussis toxin (PTX) for both receptor subtypes, while only about 50% of the internalization was blocked by PTX. The results indicate that the extent of internalization of alpha2A-AR and alpha2B-AR is proportional to agonist efficacy, but only partly dependent on Gi protein coupling.

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.

Monoclonal antibodies (Mabs) against human alpha2C2-adrenergic receptor (alpha2C2-AR) were raised in mice and characterized. Bacterially expressed fusion protein consisting a sequence from the putative third intracellular loop (amino acids 213-343) of human alpha2C2 and glutathione-S-transferase (GST) was used as antigen. Results from mass spectrometry of purified thrombin cleaved alpha2C2 polypeptide suggested that the epitope region would lie near the aminoterminal end of the 3rd intracellular loop of human alpha2C2-AR. Elevation of Mabs was detected with Western blotting from mouse blood samples. Three alpha2C2 specific cell clones were expanded to in vitro production in hollow fiber systems. The specificity of the Mabs was further determined by immunoprecipitation and immunocytochemistry. Scatchard analysis of thrombin digested, purified, Europium-labelled antigen (amino acids 213-343 of alpha2C2) revealed binding affinity constants of 0.4 x 10(9), 0.7 x 10(9) and 1.6 x 10(9) M(-1) and Kds of 2.6, 1.4 and 0.6 nM for the three Mabs 2B1, 3G3 and 7G1, respectively.