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Determination of Alkali-Sensing Parts of the Insulin Receptor-Related Receptor Using the Bioinformatic Approach.
Deyev IE
,
Popova NV
,
Petrenko AG
.
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IRR (insulin receptor-related receptor) is a receptor tyrosine kinase belonging to the insulin receptor family, which also includes insulin receptor and IGF-IR receptor. We have previously shown that IRR is activated by extracellular fluid with pH > 7.9 and regulates excess alkali excretion in the body. We performed a bioinformatic analysis of the pH-sensitive potential of all three members of the insulin receptor family of various animal species (from frog to man) and their chimeras with swapping of different domains in the extracellular region. An analysis using the AcalPred program showed that insulin receptor family proteins are divided into two classes: one class with the optimal working pH in the acidic medium (virtually all insulin receptor and insulin-like growth factor receptor orthologs, except for the IGF-IR ortholog from Xenopus laevis) and the second class with the optimal working pH in the alkaline medium (all IRR orthologs). The program had predicted that the most noticeable effect on the pH-sensitive property of IRR would be caused by the replacement of the L1 and C domains in its extracellular region, as well as the replacement of the second and third fibronectin repeats. It had also been assumed that replacement of the L2 domain would have the least significant effect on the alkaline sensitivity of IRR. To test the in silico predictions, we obtained three constructs with swapping of the L1C domains, the third L2 domain, and all three domains L1CL2 of IRR with similar domains of the insulin-like growth factor receptor. We found that replacement of the L1C and L1CL2 domains reduces the receptor's ability to be activated with alkaline pH, thus increasing the half-maximal effective concentration by about 100%. Replacement of the L2 domain increased the half-maximal effective concentration by 40%. Thus, our results indicate the high predictive potential of the AcalPred algorithm, not only for the pH-sensitive enzymes, but also for pH-sensitive receptors.
Fig. 1.
A – Sequence analysis of insulin receptor family
ectodomains using the AcalPred program. The relative probabilities of
activation of insulin receptor family ectodomains from various species at
alkaline pH are shown
in table.
Full names of the species are given in the
Experimental section. B – The graphical representation
of the aforedescribed probabilities. The red line highlights the notional
boundary value prediction – 0.5. Proteins with a predicted probability of
alkaline sensitivity greater than 0.5 are shown as “alkaline” (in
the red zone), and proteins with the probability less than 0.5 are indicated as
“acidic” proteins (in the blue zone)
Fig. 2.
A – Schematic representation of the resulting chimeric
proteins. IRR domains are shown in white; IR, in striped; and IGF-IR domains,
in gray. L1 and L2 – L-domains, C – furin-like cysteine-rich
domain, FnIII-1 and FnIII 2 & 3 – the first or second and third
fibronectin repeats. B – Sequence analysis of the
ectodomains of the chimeric receptors described above using the AcalPred
program. The relative predicted probabilities that the ectodomain is an
“alkaline” protein are shown
in Table.
Predicted probability values
above 0.5 are shown in red (“alkaline” proteins), and those less
than 0.5 are shown in blue (“acidic” proteins)
Fig. 3.
A – Activation of chimeric receptors at alkaline pH.
HEK293 cells after expression of chimeric proteins were treated with 60 mM
Tris-HCl buffers with pH 7.3 or 9.0, then lysed, and the proteins were
separated by SDS-PAGE and transferred onto nitrocellulose membranes for Western
blot analysis. An antibody to the phosphorylated IRR was used to detect
phosphorylated receptors; the beta subunit was detected with an antibody
against the C-terminal part of the IRR. B – pH-dependent
activation curves of IRR and chimeric receptors. HEK293 cells after expression
of chimeric proteins were treated with a buffer with pH ranging from 7.3 to 9.4
(7.3; 7.8; 8.0; 8.2; 8.5; 8.7; 9.0; 9.2; 9.4); the cells were then lysed, and
the proteins were separated by SDS-PAGE and transferred onto a nitrocellulose
membrane for Western blot analysis. Antibody to the phosphorylated IRR was used
for detecting phosphorylated receptors; the beta-subunit was detected with an
antibody against the C-terminal part of the IRR. The phosphorylated receptor
was normalized to the total amount of the receptor (signal from beta-receptor
subunits) for each pH value. The normalized signals for each pH (n ≥ 3)
were calculated using the GraphPad Prism 5 software with One site –
Specific binding with Hill slope interpolation. On each plot, the Y axis shows
the percentage of the maximum average activation at pH 9.4
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