Absence of functional insulin receptor substrate-3 (IRS-3) gene in humans.
OBJECTIVE
Insulin receptor substrate (IRS) proteins play important roles in insulin action and pancreatic beta-cell function. At least four mammalian IRS molecules have been identified. Although genes and cDNAs encoding human IRS-1, IRS-2, and IRS-4 have been cloned, IRS-3 has been identified only in rodents. Thus, we have attempted to clone the human IRS-3 gene.


METHODS
Insulin-stimulated rat or human adipocytes were subjected to Western blot analysis to assess IRS-3 tyrosine phosphorylation. Human liver and adipose cDNA libraries were screened in an effort to clone IRS-3 cDNA. A PCR-based approach was designed to amplify IRS-3 cDNA. Reverse transcription PCR was carried out using mRNA from adipose tissue, liver, and skeletal muscle as templates in combination with an in silico screen using mouse IRS-1, IRS-2 and IRS-3 in a tblastn search of the draft public human genome.


RESULTS
In human adipocytes we did not detect a M(r) 60 000 phosphoprotein corresponding to IRS-3, whereas in rat adipocytes IRS-3 protein and insulin-stimulated tyrosine phosphorylation was readily observed. None of the molecular approaches provided evidence for a functional IRS-3gene in human tissue. Two deletions in human IRS-3 gene were identified using bioinformatics. The human IRS-3 gene product is predicted to lack a phosphotyrosine binding domain and also the sequence corresponding amino acid 353-407 of murine IRS-3. The contiguous sequence of genomic DNA between these two homologous regions does not have the coding information for human IRS-3.


CONCLUSIONS
In silico screening of the human IRS-3 genome region, combined with further biological and molecular validation, provides evidence against a functional IRS-3 in humans.
Björnholm
M
M
Integrative Physiology at Karolinska Institute, Stockholm, Sweden.
He
A R
AR
Attersand
A
A
Lake
S
S
Liu
S C H
SC
Lienhard
G E
GE
Taylor
S
S
Arner
P
P
Zierath
J R
JR
D017667:Adipocytes / Q000378:metabolism; D000328:Adult; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D003001:Cloning, Molecular; D019295:Computational Biology; D005260:Female; D017353:Gene Deletion; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D008297:Male; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology; D010766:Phosphorylation; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014443:Tyrosine / Q000378:metabolism
C491585:IRS3P protein, human; D055504:Insulin Receptor Substrate Proteins; C108872:Irs3 protein, mouse; C491584:Irs3 protein, rat; D010750:Phosphoproteins; D014443:Tyrosine