Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

The complete nucleotide sequence of the I-E alpha d immune response gene.

We have isolated and sequenced the complete murine I-E alpha immune response gene of the H-2db haplotype. The I-E alpha d gene consists of 5300 basepairs and is organized into five or possibly six exons that correspond to different domains of the alpha chain. The amino acid sequence deduced from the I-E alpha gene shows 75% homology to its human counterpart, the HLA-DR alpha chain. The absence of I-E antigen in H-2 mice is due to lack of E alpha chain synthesis. We show here that this defect is caused by a deletion in the 5' end of the I-E alpha b gene.     

Localization of neuropeptide Y receptor Y5 mRNA in the guinea pig brain by in situ hybridization

Neuropeptide Y (NPY) has prominent stimulatory effects on food intake in virtually all animals that have been studied. In mammals, the effect is primarily mediated by receptors Y1 and Y5, which seem to contribute to different aspects of feeding behavior in guinea pigs and rats/mice. Interestingly, differences in receptor distribution among mammalian species have been reported. To get a broader perspective on the role of Y5, we describe here studies of guinea pig (Cavia porcellus), a species which due to its phylogenetic position in the mammalian radiation is an interesting complement to previous studies in rat and mouse. Guinea pig brain sections were hybridized with two 35S-labeled oligonucleotides complementary to Y5 mRNA. The highest expression levels of Y5 mRNA were observed in the hippocampus and several hypothalamic and brain stem nuclei implicated in the regulation of feeding, such as the paraventricular, arcuate and ventromedial hypothalamic nuclei. This contrasts with autoradiography studies that detected low Y5-like binding in these areas, a discrepancy observed also in rat and human. Y5 mRNA expression was also seen in the striatum, in great contrast to mouse and rat. Taken together, these data show that Y5 mRNA distribution displays some interesting species differences, but that its expression in feeding centers seems to be essentially conserved among mammals, adding further support for an important role in food intake.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Pharmacological comparison of rat and human melanocortin 3 and 4 receptors in vitro

The melanocortin 3 and 4 receptors are G-protein-coupled receptors found in the hypothalamus with important role in regulation of the energy balance. In this study, we performed pharmacological comparison of the rat and human melancortin (MC) 3 and MC4 receptors. We transiently expressed the genes for these receptors individually in a mammalian cell line and determined the binding affinities to several MSH peptides. The results showed no major difference between the rat and human MC3 receptors while the rat MC4 receptor had higher affinity to several peptides compared with the human MC4 receptor. NDP-, alpha-, beta-, gamma-MSH, ACTH(1-24), HS014 and MTII had from 5- to 34-fold higher affinity for the rat MC4 receptor, while SHU9119, HS024 and HS028 had similar affinity for both the MC4 receptors. Pharmacological species difference have earlier been reported for the MC1 and MC5 receptors but this is the first report showing important differences between the rat and human MC4 receptors.     

Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands

The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, receptor binding, and anatomical distribution. We have investigated the differences in binding profile between the cloned human, rat, and guinea pig Y4 receptors using NPY analogues with single amino acid replacements or deletion of the central portion. The most striking result was the increase in affinity for the rat receptor, but not for human or guinea pig, when amino acid 34 was replaced with proline; [Ahx(8-20),Pro(34)]NPY bound to the rat Y4 receptor with 20-fold higher affinity than [Ahx(8-20)]NPY. Also, the rat Y4 tolerates alanine in position 34 since p[Ala(34)]NPY bound with similar affinity as pNPY while the affinity for hY4 and gpY4 decreased about 50-fold. Alanine substitutions in position 33, 35, and 36 as well as the large loop-deletion, [Ahx(5-24)]NPY, reduced the binding affinity to all three receptors more than 100-fold. NPY and PYY competed with (125)I-hPP at Y4 receptors expressed in CHO cells according to a two-site model. This was investigated for gpY4 by saturation with either radiolabeled hPP or pPYY. The number of high-affinity binding-sites for (125)I-pPYY was about 60% of the receptors recognized by (125)I-hPP. Porcine [Ala(34)]NPY and [Ahx(8-20)]NPY bound to rY4 (but not to hY4 or gpY4) according to a two-site model. These results suggest that different full agonists can distinguish between different active conformations of the gpY4 receptor and that Y4 may display functional differences in vivo between human, guinea pig, and rat.