2002 Global Researcher Conference Proceeding
April 26 - 28, 2002
| Conference: | 2002 Global Researcher Conference |
|---|---|
| Title: | Comparative analysis of human and murine vasopressin V2 receptor genes and their encoded proteins reveal striking differences in their genomic structure and functional properties |
| Authors: | Leder, Gabriele; Platzer, Matthias; Valet, Susanne; Krause, Eberhard; Rosenthal, Walter; Oksche, Alexander |
| Institutions: | Forschungsinstitut fur Molekulare Pharmakologie, Institut fur Molekulare Biotechnologie Jena, Charite - Universitatsmedizin Berlin, Institut Fuer Pharmakologie |
In the course of an international comparative sequencing effort of the orthologous regions of human Xq28 and mouse XB, we isolated and sequenced two PAC clones that span a genomic region of 110,020 bp. This region contains the complete murine genes L1cam, Avpr2, c1, Te2, Renbp and the 5' region of the Hcfc1 gene. The untranscribed regions between Avpr2 and the proximal L1cam and the distal c1 cover 11,516 bp and 104 bp respectively. Comparative analysis of this locus with its human orthologue (official gene symbol AVPR2) revealed a high degree of conservation: both gene structures and gene orientations are identical and intron sizes and intergenic distances are closely related. Despite a high conservation between the orthologous regions XB in mouse and Xq28 in man, a lack of intergenic homology, in particular in the 5’ UTR of the V2 receptor genes, was found. Only 680 bp immediately upstream of the start codon show significant homology (56% identity).
The murine (mV2R) and human vasopressin V2 receptor (hV2R) coding regions display a high degree of identity in the nucleotide (84%) and amino acid sequence (88%). Expression of both receptors in CHO and COS.M6 cells, however, revealed marked differences. The mV2R displayed a higher affinity for [3H]AVP and lower halfmaximal activation rate for AVP-mediated stimulation of the adenylyl cyclase than the human orthologue (Kd values were 0.4 +/- 0.2 nM vs. 2.2 +/- 1.1 nM and EC50 values were 0.6+/- 0.3 nM and 3.3 +/- 2.0 for mV2R and hV2R respectively). Transient expression of the mV2R yielded lower maximal binding capacities which reached only 20 to 30 % of the levels obtained for the hV2R. Laser scanning microscopy of HEK 293 cells transiently expressing the hV2R/GFP fusion protein showed strong expression within the plasma membrane, whereas the mV2R/GFP fusion protein was mainly located intracellularly within the endoplasmic reticulum (ER).
A chimera of the hV2R, in which the first and second extracellular loop (ECL) was replaced by the corresponding ECL’s of the mV2R, displayed similar functional properties (Kd , Bmax) as the mV2R. Exchange of only the first ECL was responsible for the reduction in maximal binding capacity, whereas only the combination of both ECL‘s resulted in the maximal increase in ligand affinity. The exchange of lysine 100 by aspartate (corresponding amino acid in the mV2R) in the first ECL of hV2R was sufficient to reduce cell surface expression which was accompanied by retention within the ER. In the reverse experiment, the exchange of aspartate 100 by lysine in the mV2R caused an increase in the cell surface expression and resulted in predominant plasma membrane localization. Thus a single amino acid in the first ECL, variant between mV2R and hV2R, determines the efficiency of cell surface expression. Species-dependent differences in the affinity to [3H]AVP can be attributed to variant amino acids within the first and second ECL.
Oksche, et al., compared the vasopressin-2 receptor (V2R) genes and the encoded V2R proteins of man and mouse. Their analysis revealed significant differences in both the structure of the respective genes and the function of the two proteins.
The coding regions of the murine and human V2R gene are similar, but not the promoter regions. The latter are of importance for the regulation of gene expression. Thus, differences in the regulation of V2R expression between man and mouse are likely. The proteins generated from these genes, the human V2R (hV2R) and the murine (mouse) V2R (mV2R), have similar amino acid sequences. Yet analysis shows that they express quite differently in laboratory cell cultures. mV2R has a higher affinity for vasopressin (VP) and a higher efficiacy to stimulate adenylyl cyclase in the presence of VP, but shows a lower binding capacity than hV2R. hV2R, when fused with GFP was found within the cell membrane, whereas, the mV2R/GFP fusion protein was found in the cell interior in a section of the cell called the endoplasmic reticulum.
The researchers experimented with the structures of the hV2R and mV2R by replacing specific sections of hV2R with the corresponding section of mV2R and vice versa. They found that the difference in affinity to AVP and the cell surface expression by hV2R and mV2R can be attributed to differences in the amino acids that occur in two specific sections of the V2R, the first and second extracellular loops.