Biomedical subjects

P A Frändberg

Publications and source records attributed to P A Frändberg.

7 recordsLinked to original sources

PubMed2001

Cysteine residues are involved in structure and function of melanocortin 1 receptor: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist.

Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.

Adrenocorticotropic Hormone↗

PubMed1998

Human pigmentation phenotype: a point mutation generates nonfunctional MSH receptor.

alpha-Melanocyte stimulating hormone (alpha-MSH) regulates skin and hair pigmentation by modulating the activity of MSH receptor (MC1R). We have identified Arg151Cys variant of human MC1R in genomic DNA isolated from a person with red hair and light skin of type I. The Arg151Cys variant of MC1R binds to radio-labelled analogue of alpha-MSH with identical affinity as wild type MC1R but can not be stimulated to produce cyclic AMP (cAMP). The mutation Arg151Cys renders human MC1R completely nonfunctional, which explains the red hair, light skin and poor tanning ability (skin type I). This is the first report ever describing a nonfunctional MC1R isolated from a human subject.

Animals↗

PubMed1998

Amino acid residues in third intracellular loop of melanocortin 1 receptor are involved in G-protein coupling.

To delineate domains essential for G-protein coupling in melanocortin 1 receptor (MC1R), we mutated polar and basic residues to alanine at eleven positions in the putative third intracellular loop and determined consequent changes in the ligand binding and generation of second messenger cAMP. Results demonstrate that ligand binding affinity was not affected by any of the mutations. However, every mutant displayed reduced functional response as compared to the wild type receptor. Replacement of residues (K226, R227, Q228, R229, H232, Q233 and K238) present in second half of third intracellular loop resulted in an almost complete loss of functional response. The results have demonstrated that the amino acid residues present in C-terminal portion of third intracellular loop of MC1R are involved in coupling to G-protein and that a region of four amino acids, K226-R227-Q228-R229 is essential for coupling of MC1R to G-protein.

Amino Acid Sequence↗

PubMed1997

Glutamine235 and arginine272 in human melanocortin 5 receptor determines its low affinity to MSH.

The human melanocortin 5 receptor (hMC5R) in the melanocortin receptor family has been identified as the receptor with low affinity towards alpha-MSH. Here we show that the glutamine at position 235 and arginine at the position 272 in the hMC5R are contributing to the low affinity of this receptor. Glutamine235 and arginine272 in hMC5R were mutated to lysine (Q235K) and cysteine (R272C), respectively, residues which are conserved at these positions in other melanocortin receptor subtypes. Upon these mutations affinity of alpha-MSH for hMC5R was increased 10-fold for Q235K and 690-fold for R272C mutants, respectively. The results explain the unusually low affinity of the hMC5R to the melanocortic ligands and suggest the importance of these conserved residues in maintaining the high affinity form of melanocortin receptors.

Amino Acid Sequence↗

PubMed1995

A three dimensional model for the interaction of MSH with the melanocortin-1 receptor.

A model for the interaction of the melanocortin-1 (MC1) receptor with MSH peptide has been constructed. The model was built by homology modelling using bacteriorhodopsin as template. A cyclic analogue of MSH could be docked into a binding pocket located between transmembrane (TM) domains 2, 3 and 6 of the receptor. The most significant receptor to ligand interactions occur between D117 in TM3 of receptor with histidine in cyclic MSH-peptide, H260 in TM6 with glutamic in peptide and D121 in TM3 with arginine in peptide. The model finds support from earlier mutagenesis data.

Amino Acid Sequence↗

PubMed1994

Evidence for alternate points of attachment for alpha-MSH and its stereoisomer [Nle4, D-Phe7]-alpha-MSH at the melanocortin-1 receptor.

The molecular basis for the alpha-melanocyte stimulating hormone (alpha-MSH) stereoselectivity was studied by examining ligand binding to site specific mutants of the melanocortin 1 receptor (MC1R). The amino acids Asp117, Phe179, His209 and His260 were targeted for mutation to alanine as they are conserved in the melanocortin receptor family. Expression of the wild type and the MC1R mutants in COS-7 cells revealed that the binding affinities for the alpha-MSH (L-isomer) was reduced by 267 fold for the D117-->A mutant and 132 fold for the H260-->A mutant. In contrast, the binding affinity for the [Nle4, D-Phe7]-alpha-MSH (NDP-MSH; D-isomer) remain unchanged between the wild type and the mutants. Moreover, the mutants also displayed reduction in affinity to L-isomers of all the other melanocortic peptides examined. Thus, the mutation of single amino acids in the third and sixth transmembrane segments results in the display of ligand stereoselectivity of the MC1R. In addition, these data represent the first evidence of the different binding epitopes on a G-protein coupled receptor for a peptide ligand stereoisomers, both of which are shown to be potent agonists.

Amino Acid Sequence↗

PubMed1991

Heterogeneity in human soluble guanylate cyclase due to alternative splicing.

Two forms of the smaller subunit of the human soluble guanylate cyclase enzyme have been cloned by using PCR. One of the clones (HSGC-1) is identical to bovine and rat lung smaller subunit cyclase. However, the other (HSGC-2) is lacking 33 amino acids. Comparison of its sequence with published partial genomic sequences of bovine guanylate cyclase indicates that HSGC-2 is formed due to alternative splicing.

Animals↗