Question

Suppose SCAFF A is a scaffold protein for vitamin A.    Would likely happen if the SCAFF A protein were mutated and impaired? How would this affect the individual?

Answer 1

Protein scaffolds are members of the signaling cascade downstream of cell surface receptors. Scaffold proteins help relay the message between the cell membrane and nucleus faster. They do this by serving as a docking site for multiple protein partners in the cascade so they can be near each other.

The Rules of Protein Structure. The function of a protein is determined by its shape. The shape of a protein is determined by its primary structure (sequence of amino acids). The sequence of amino acids in a protein is determined by the sequence of nucleotides in the gene (DNA) encoding it.

A recurring theme is that scaffold proteins increase the flexibility of a cell's signaling responses. Scaffold proteins can serve as targets for many forms of regulatory modulation, which allows the cell to achieve a wide range of behaviors from a limited set of components.

What are Scaffold Proteins?

Protein scaffolds are members of the signaling cascade downstream of cell surface receptors. Scaffold proteins help relay the message between the cell membrane and nucleus faster. They do this by serving as a docking site for multiple protein partners in the cascade so they can be near each other. This proximity cuts down the time required for one protein in the cascade to find its partner. Some protein scaffolds remain unloaded until a message from an activated membrane receptor reaches them, after which they are docked by several proteins in the cascade. Other protein scaffolds are docked by proteins in the cascade even before an activated membrane receptor sends a message to them, increasing the efficiency in which the message is relayed from receptor to nucleus.

GPCRs and Scaffold Proteins

G-protein Coupled Receptors (GPCRs) are a large class of cell surface proteins that relay extracellular signals to the nucleus. An example of a GPCR is the β-adrenergic receptor that senses the hormone adrenaline. Once activated by a ligand at the outer cell surface, GPCRs activate their cognate G-proteins that reside on the inner cell membrane. The activated G-proteins then slide along the inside of the cell membrane to activate a cascade of proteins and enzymes that in turn amplify the original message received by the GPCR. Scaffold proteins are key hubs of information downstream of activated GPCRs. Scaffold proteins recruit downstream members of a signaling cascade to the inner cell membrane very quickly, or ahead of time, making it efficient for the message to move from GPCR to cytosol.

Activation States of the Signaling Cascade

The complexity of intracellular signaling is that the proteins in a signaling cascade have varying states of activity (i.e. off, partially active, fully active). These states depend on conformational changes that result from interactions with other proteins. They also depend on the presence or absence of moieties, such as phosphate groups, ubiquitin groups, and calcium ions. The activation state of a signaling protein is often used as an indicator of whether a certain pathway has been activated. For example, phosphorylated MEK or ERK is often interpreted as representing activation of an intracellular signaling pathway downstream of a GPCR. Furthermore, these signaling proteins can have multiple sites of phosphorylation, different combinations of which affect the level of the protein’s activity or half-life. Proteins such as MEK and ERK are known to bind near each other on scaffold proteins. The ability scaffold proteins to bind MEK and ERK is also regulated in terms of the scaffold’s activation state, which changes after post-translational modification by addition of chemical moieties.

Picking the Right Epitopes for Accurate Information

The aforementioned complexity of intracellular signaling is precisely why picking the right antibodies is crucial when researchers seek to accurately determine the state of a cell in response to extracellular stimuli. Each active conformation of a protein or phosphorylation site can serve as a different epitope that can be bound by an antibody. Thus, antibodies that are designed to detect the presence or absence of a chemical moiety on a signaling protein can give researchers a clear picture of what pathway has been activated and to what extent. Phosphorylated epitopes are often targets of immunoblotting and in situ immunolocalization assays. Since a protein can have multiple phosphorylation sites, each of which affects the protein differently, in addition to multiple sites for moieties such as ubiquitination, picking the right epitopes to detect is a fruitful – but nontrivial – task.

Multiple epitopes, whether on the same protein or different proteins, can be simultaneously or sequentially detected on the same blot or same cells. It is thus important that the right host species for primary and secondary antibodies are considered with forethought. Single domain antibodies are also useful for these experiments, which add to the versatility of combinations of antibodies that can be used in the same assay.

Both monoclonal and polyclonal antibodies are useful in detecting the state of intracellular signaling cascades. Our antibody selection guide makes picking the right combination of antibodies easy. It takes into consideration the specimen species, the host species, the desired level of specificity, and cross-reactivity concerns, among other factors. The antibody selection guide will lead you to the best options for single-purpose antibodies or those that you will use in multiple applications. The right combination of antibodies targeted to strategic epitopes will bring clarity to the state of any intracellular signaling cascade.